Carbonic anhydrase targeting agents and methods of using same

ABSTRACT

The invention provides agents that target carbonic anhydrase, which can be used as imaging agents or therapeutic agents. The agents can be used to image tumor hypoxia as well as other physiological processes in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/467,694, filed May 9, 2012, which claims the benefit of and priorityto U.S. Provisional Patent Application No. 61/483,979, filed May 9,2011, the contents of each of which are hereby incorporated byreference.

FIELD OF THE INVENTION

The invention provides agents that target carbonic anhydrase, which canbe used as imaging agents or therapeutic agents. The agents can be usedto image tumor hypoxia as well as other physiological processes in asubject.

BACKGROUND

Current approaches for assessing molecular endpoints in certain diseasesusually require tissue and blood sampling, surgery, and in the case ofexperimental animals, sacrifice at different time points. Despiteimprovements in noninvasive imaging, more sensitive and specific imagingagents and methods are urgently needed. Imaging techniques capable ofvisualizing specific molecular targets and/or entire pathways wouldsignificantly enhance our ability to diagnose and assess treatmentefficacy of therapeutic interventions for many different disease states.Most current imaging techniques report primarily on anatomical orphysiological information (e.g., magnetic resonance imaging (MRI),computed tomography (CT), and ultrasound). Newer modalities such asoptical imaging and new molecular imaging probes have the potential torevolutionize the way disease is detected, treated, and monitored.

In particular, optical imaging offers several advantages that make it apowerful molecular imaging approach, both in the research and clinicalsettings. Specifically, optical imaging can be fast, safe, costeffective and highly sensitive. Scan times are on the order of secondsto minutes, there is no need for ionizing radiation, and the imagingsystems can be relatively simple to use. In addition, optical probes canbe designed as dynamic molecular imaging agents that may alter theirreporting profiles in vivo to provide molecular and functionalinformation in real time. In order to achieve maximum penetration andsensitivity in vivo, the choice for most optical imaging in biologicalsystems is within the red and near-infrared (NIR) spectral region(600-900 nm), although other wavelengths in the visible region can alsobe used. In the NIR wavelength range, absorption by physiologicallyabundant absorbers such as hemoglobin or water, as well as tissueautofluorescence, is minimized.

Hypoxia, or hypoxiation, is a pathological condition in which the bodyas a whole (generalized hypoxia) or a region of the body (tissuehypoxia) is deprived of adequate oxygen supply. Variations in arterialoxygen concentrations can be part of the normal physiology, for example,during strenuous physical exercise. A mismatch between oxygen supply andits demand at the cellular level may result in a hypoxic condition.

Tumor hypoxia is the situation where tumor cells have been deprived ofoxygen. As a tumor grows, it rapidly outgrows its blood supply, leavingportions of the tumor with regions where the oxygen concentration issignificantly lower than in healthy tissues. It can also be a result ofthe high degree of cell proliferation undergone in tumor tissue, causinga higher cell density, and thus taxing the local oxygen supply.

The carbonic anhydrases (or carbonate dehydratases) form a family ofenzymes that catalyze the rapid interconversion of carbon dioxide andwater to bicarbonate and protons (or vice-versa), a reversible reactionthat occurs rather slowly in the absence of a catalyst. One of thefunctions of the enzyme in animals is to interconvert carbon dioxide andbicarbonate to maintain acid-base balance in blood and other tissues,and to help transport carbon dioxide out of tissues.

Carbonic anhydrases (CAs) are a large family of zinc metalloenzymes thatparticipate in a variety of biological processes, including respiration,calcification, acid-base balance, bone resorption, and the formation ofaqueous humor, cerebrospinal fluid, saliva, and gastric acid. They showextensive diversity in tissue distribution and in their subcellularlocalization. CA IX is a transmembrane protein that has been shown to besignificantly upregulated under hypoxic conditions and plays a criticalrole, along with the intracellular carbonic anhydrase II, in hypoxiaassociated extracellular acidifcation. It is expressed in all clear-cellrenal cell carcinoma, but is not detected in normal kidney or most othernormal tissues. It may be involved in cell proliferation andtransformation. Importantly, tumor hypoxia and subsequent expression ofCA IX are associated with poor prognosis and treatment outcomes innumerous cancer types. The ability to more accurately and efficientlydetect and quantify carbonic anhydrase-associated hypoxia will aid inthe understanding of biological phenomena such as cellular proliferationand cancer, as well as in the determination of the most appropriatetreatment regimens.

SUMMARY OF THE INVENTION

The invention provides fluorescent imaging agents that bind to carbonicanhydrase, in particular carbonic anhydrase IX (CA IX), and can be usedin a variety of in vitro and in vivo applications, including but notlimited to the identification of hypoxic cells such as would be found inhypoxic tumors. The invention also provides CA IX agents/ligands thatare fluorescent in the far-red or near-infrared region that are ofparticular utility for in vivo imaging of carbonic anhydrase in liveanimals. In addition, the invention further provides agents that,independently, contain a far-red or near-infrared fluorophore that hasbeen modified by a plurality of chemical modifying groups that can beused for optimization of in vitro and in vivo properties of the agent.

In one aspect, the carbonic anhydrase targeting agent comprises: acarbonic anhydrase binding moiety comprising a sulfonamide moietyoptionally substituted with an aliphatic, aromatic or heteroaromaticmoiety; and a fluorescent reporter chemically linked, optionally througha linker (L) moiety to the carbonic anhydrase moiety wherein thefluorescent moiety optionally further comprises a plurality of chemicalmodifying groups.

In another aspect, the invention comprises: (a) a carbonic anhydrasebinding (CAB) moiety comprising a sulfonamide group and an aromatic orheteroaromatic moiety; (b) a far-red or near-infrared fluorophore (F)chemically linked, optionally through a linker (L) to the CAB moiety;and (c) a plurality of chemical modifiers (M), each independently andoptionally linked to the fluorophore through a linker (L).

The chemical modifying moieties (M) can include, for example, two toeight individual modifying groups, each chemically linked to thefluorophore. Agents can comprise CAB, a fluorophore, and a plurality ofmodifying groups.

In certain embodiments, the carbonic anhydrase targeting agent isrepresented by formula (I), or a salt thereof:

-   -   wherein:    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety;    -   Q is a substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl,        alkoxy, or thioalkyl group; or Q is absent;    -   L₁, L₂, and L₃ each represent independently for each occurrence        a bond or a linker moiety;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, C(CH₂Y₁)(CH₂Y₂), O, S,        or Se;    -   Y₁ and Y₂ are independently hydrogen or a C₁-C₂₀ aliphatic        group, each of which is optionally substituted with L₃-M;    -   M, independently for each occurrence, is hydrogen or a chemical        modifying moiety.

In certain embodiments, the carbonic anhydrase targeting agent isrepresented by formula (II), or a salt thereof:

-   -   wherein:    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic,    -   aromatic or heteroaromatic moiety;    -   Q is a substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl,        alkoxy, or thioalkyl group; or Q is absent;    -   L₁, L₂, and L₃ each represent independently for each occurrence        a bond or a linker moiety;    -   R is, independently for each occurrence, hydrogen, substituted        or unsubstituted aryl, substituted or unsubstituted heteroaryl,        C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group, each        of which is optionally substituted with L₃-M;    -   n is 1, 2, or 3;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, C(CH₂Y₁)(CH₂Y₂), O, S,        or Se;    -   Y₁ and Y₂ are independently hydrogen or a C₁-C₂₀ aliphatic        group, each of which is optionally substituted with L₃-M; and    -   M, independently for each occurrence, is hydrogen or a chemical        modifying moiety.

In certain embodiments, the carbonic anhydrase targeting agent comprisesa compound of formula (III) or a salt thereof:

F-L-CAB,  (III)

-   -   wherein    -   F is a near infrared fluorochrome;    -   L is an optional linker; and    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety.

In certain embodiments, the chemical modifying moiety, M is selectedfrom hydrogen, alcohol, sulfonic acid, sulfonate, sulfonamide,sulfoxide, sulfone, carboxylate, ketone, phosphonate, phosphate;iminodiacetate, ethylenediamine tetraacetic acid, diethylenetriaminepentaacetic acid, tetraazacyclododecane tetraacetic acid, an amino acid,polyamino acid, oligo- or polyethylene glycol, amine, quaternaryammonium ion, glucosamine, galactosamine, or mannosamine. In otherembodiments, the chemical modifying moiety, M is a carboxylate,phosphonate, phosphate, or iminodiacetate. In other embodiments, thechemical modifying moiety, M is a sulfonate.

In certain embodiments, certain M modifications of the compounds of thepresent invention result in fluorescent CA imaging agents that havehigher selectivity for CA IX (relative to a CA other than CA IX (such asCA II)) than the CAB binding group not linked to the chemically modifiedfluorophore.

In certain embodiments, certain M modifications of the compounds of thepresent invention result in fluorescent CA imaging agents that do notnonspecifically cross cell membranes, thus enabling even greaterselectivity for cell surface CAs over cytoplasmic CAs, such as CA II.

In certain embodiments, certain M modifications of the compounds of thepresent invention result in fluorescent CA imaging agents that have lownonspecific accumulation in animal tissues that are CA IX negative, thusenabling high contrast with respect to adjacent tissues that are CA IXpositive.

In certain embodiments, the chemical modifying moiety, M, modifies thecarbonic anhydrase targeting agent with a net negative charge rangingfrom −3 to −12 at neutral pH.

In certain embodiments, the chemical modifying moiety, M, reduces thenonspecific cell membrane permeability of the carbonic anhydrasetargeting agent. Furthermore, in other embodiments, the chemicalmodifying moiety, M, reduces the nonspecific tissue accumulation of thecarbonic anhydrase targeting agent when administered to a live animal.

In certain embodiments, the bond or linker moiety, L, comprises a moietyselected from the group consisting of glycine, alanine, β-alanine,—NH—(CH₂)_(n)—C(═O)— where n=1-8, 4-aminomethylbenzoic acid, cysteicacid, glutamic acid, amino-polyethylene glycol-carboxylic acid,amino-polyethylene glycol amine, ethylenediamine, propylenediamine,spermidine, spermine, hexanediamine, and diamine-amino acids, such ashomolysine, lysine, ornithine, diaminobutyric acid and diaminopropionicacid, succinic acid, glutaric acid, suberic acid, or adipic acid.

In certain embodiments, the carbonic anhydrase targeting moiety (CAB) informula I, II or III is one of the following:

In certain embodiments, the carbonic anhydrase imaging agent is selectedfrom the group consisting of:

-   3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N-(4-oxo-4-((4-sulfamoylbenzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((4-sulfamoylbenzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-1-(4-sulfamoylphenyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-2-azaheptaheptacontan-77-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N-(4-oxo-4-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indol-3-ium-6-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-1-(4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-2-azaheptaheptacontan-77-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;    and-   3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1-sulfonate;    and pharmaceutically acceptable salts thereof.

Another aspect of the invention provides pharmaceutical compositionsuitable for administration to a subject, the pharmaceutical compositioncomprising a carbonic anhydrase targeting agent and a pharmaceuticallyacceptable excipient.

In certain embodiments, the invention provides a method of in vivoimaging, the method comprising: (a) administering to a subject acarbonic anhydrase targeting agent; (b) allowing the agent to distributewithin the subject; and (c) detecting a signal emitted by the carbonicanhydrase targeting agent. In certain embodiments, steps (a)-(c) arerepeated at predetermined time intervals thereby to permit evaluation ofthe emitted signals of the carbonic anhydrase targeting agent in thesubject over time.

In certain embodiments, the invention provides a method of in vivooptical imaging, the method comprising: (a) administering to a subject acarbonic anhydrase targeting agent, wherein the agent comprises afluorophore or fluorochrome; (b) allowing the agent to distribute withinthe subject; (c) exposing the subject to light of a wavelengthabsorbable by the fluorochrome; and (d) detecting a signal emitted bythe agent. Furthermore, in other embodiments, steps (a)-(d) are repeatedat predetermined time intervals thereby to permit evaluation of theemitted signals of the carbonic anhydrase agents in the subject overtime.

In certain embodiments, the subject is an animal or a human.

In certain embodiments, the signal emitted by the agent is used toconstruct an image, for example, a tomographic image.

In certain embodiments, during step (a) two or more imaging probes whosesignal properties are distinguishable from one another are administeredto a subject, wherein at least one of the imaging probes is a carbonicanhydrase targeting agent. In other embodiments, in step (a), cellslabeled with the carbonic anhydrase targeting agent are administered tothe subject. In other embodiments, the signal emitted by the carbonicanhydrase targeting agent is used to monitor trafficking andlocalization of the cells.

In other embodiments, the illuminating and detecting steps are performedusing an endoscope, catheter, tomographic system, hand-held opticalimaging system, or an intraoperative microscope.

In certain embodiments, the presence, absence, or level of emittedsignal is indicative of a disease state. In other embodiments, themethod is used to detect and/or monitor a disease. In other embodiments,the disease is selected from the group consisting of bone disease,cancer, cardiovascular disease, atherosclerosis, restinosis, cardiacischemia, myocardial reperfusion injury, environmental disease,dermatological disease, immunologic disease, inherited disease,infectious disease, inflammatory disease, metabolic disease,neurodegenerative disease, ophthalmic disease, and respiratory disease.

In certain embodiments, the invention is a method of imaging hypoxia ina subject, the method comprising the steps of: (a) administering acarbonic anhydrase targeting agent to a subject; and (b) detecting thepresence of the agent thereby to produce an image representative ofdeficient oxygen in the subject's tissues. In certain embodiments,carbonic anhydrase agent signal in a tumor is representative of ahypoxic tumor.

In certain embodiments, the invention is a method of treating a diseasein a subject comprising administering to a subject, either systemicallyor locally, a carbonic anhydrase targeting agent, wherein the agentcomprises a radiolabel that localizes in the disease area and deliversan effective dose of radiation.

In certain embodiments, the invention is an in vitro imaging method, themethod comprising: (a) contacting a sample with a carbonic anhydrasetargeting agent; (b) allowing the agent to bind to a biological target;(c) optionally removing unbound agent; and (d) detecting signal emittedfrom the agent thereby to determine whether the agent has been activatedby or bound to the biological target. In other embodiments, the sampleis a biological sample.

In certain embodiments, the carbonic anhydrase targeting agent isfluorescent in the far-red or near-infrared.

The compounds of the present invention are efficacious for theinhibition of CAs, especially CA IX, as well as for in vitro and in vivofluorescence imaging of CAs and therefore can be used for boththerapeutic and diagnostic applications.

In addition, the invention provides methods for in vitro and in vivoimaging using the fluorescent CA imaging agents. With respect to opticalin vivo imaging, the method comprises (a) administering to a subject CAagents of the invention; (b) allowing the CA agents to distribute withinthe subject; (c) exposing the subject to light of a wavelengthabsorbable by the fluorophore of the CA agent; and (d) detecting anoptical signal emitted by the CA agent. The signal emitted by the agentcan be used to construct an image. In certain embodiments, certain ofthe images are a tomographic image. Furthermore, it is understood thatthe foregoing steps can be repeated at predetermined intervals therebypermitting evaluation of the subject over time.

The carbonic anhydrase targeting agents can be formulated into apharmaceutical composition suitable for administration to a subject, forexample, an animal and/or a human subject. The pharmaceuticalcomposition can include one or more of the carbonic anhydrase agents andone or more stabilizers in a physiologically acceptable carrier.

The subject may be a vertebrate, for example, a mammal, for example, ahuman. The subject may also be a non-vertebrate (for example, C.elegans, drosophila, or another model research organism, etc.) used inlaboratory research.

The carbonic anhydrase targeting agent, can include, for example, one tofive carbonic anhydrase binding moieties (for example, from two to five,three to five, or four to five carbonic anhydrase binding moieties),each chemically linked to the imaging reporter. The agents can comprisea plurality of carbonic anhydrase binding moieties each chemicallylinked to the imaging reporter.

Imaging reporters can be chosen, for example, from a fluorophorereporter, a fluorochrome reporter, an optical reporter, a magneticreporter, a radiolabel, an X-ray reporter, an ultrasound imagingreporter or a nanoparticle-based reporter or combination. The carbonicanhydrase agent can further comprise a biological modifier chemicallylinked to the carbonic anhydrase binding moiety or to the imagingreporter.

In addition, the present invention provides methods for in vitro and invivo imaging using the carbonic anhydrase targeting agents. With respectto optical in vivo imaging, one exemplary method comprises (a)administering to a subject one or more of the foregoing carbonicanhydrase targeting agents of the invention, wherein the agents compriseone or more fluorochromes; (b) allowing the agent to distribute withinthe subject; (c) exposing the subject to light of a wavelengthabsorbable by at least one fluorochrome; and (d) detecting a signalemitted by the carbonic anhydrase targeting agent. The signal emitted bythe agent can be used to construct an image, for example, a tomographicimage. Furthermore, it is understood that the foregoing steps can berepeated at predetermined intervals, which permit evaluation of thesubject over time.

The carbonic anhydrase targeting agents can be used to measure tumorhypoxia (oxygen defficiency) or other physiological processes such ascellular proliferation in a subject. One exemplary method comprises (a)administering one or more of the foregoing carbonic anhydrase targetingagents to a subject; (b) detecting the presence of the agent(s) therebyto produce an image representative of tumors with reduced oxygenconcentration in the subject.

In each of the foregoing methods, the subject can be a vertebrate, forexample, a mammal, for example, a human. The subject also can be anon-vertebrate (for example, C. elegans, drosophila, or another modelresearch organism, etc.) used in laboratory research.

In addition, the carbonic anhydrase targeting agents can be incorporatedinto a kit, for example, a kit with optional instructions for using thecarbonic anhydrase targeting agents in in vivo or in vitro imagingmethods. The kit optionally can include components that aid in the useof the carbonic anhydrase targeting agents, for example, buffers, andother formulating agents. Alternatively, the kit can include medicaldevices that aid in the administration and/or detection of the carbonicanhydrase targeting agents to subjects.

Other features and advantages of the invention will be apparent from thefollowing figures, detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-B depict planar reflectance (FIG. 1A) and tomographic (FIG. 1B)images of bilateral HeLa tumors at 24 h after administration ofnonbinding control compound D3 (top row of each image set) and exemplarycompound C5 (bottom row of each image set)

FIG. 2 depicts microscopy images of hypoxic and normoxic HeLa cellsincubated with exemplary agents A5 and A3 with and without preincubationof the cells with acetazolamide. Also shown is media as a negativecontrol, a fluorescent, commercial CA IX antibody as a positive control,and the nonbinding control agent D3.

FIG. 3 depicts quantification of HeLa cell fluorescent signal undernormoxic and hypoxic conditions upon incubation with nonbinding controlcompound D3 and exemplary agents A5 and A3, with and withoutacetazolamide blocking.

FIG. 4 depicts the biodistribution of nonbinding control agent D3 andexemplary agent C5 24 hours post injection in tumor bearing mice.

FIGS. 5A-B depict tomographic images (FIG. 5A) and total tumorfluorescence as quantified by FMT (FIG. 5B) in HeLa tumor bearing micewith and without pretreatment with i.v. acetazolamide.

FIGS. 6A-B depict fluorescence images of excised HeLa tumors from micetreated with exemplary agent C5 and placed in normal air or 8% oxygen(FIG. 6A). Quantitative analysis of the imaged tumors indicates agreater uptake of the exemplary agent C5 in tumors from mice underhypoxic conditions (FIG. 6B).

FIG. 7 depicts the distribution of the fluorescent carbonic anhydrasebinding compounds in tumor tissue by fluorescence microscopy. Inseparate tumor sections, the exemplary agent C5 co-localizes with bothCA IX antibody and the hypoxia marker pimonidazole (pimo).

DETAILED DESCRIPTION

The invention is based, in part, upon the discovery that it is possibleto produce stable, biocompatible fluorescent agents that target carbonicanhydrase (CA), exhibit low nonspecific cellular uptake in vitro and lownonspecific tissue uptake in vivo, and can be used in a variety of invitro and in vivo assays and imaging applications, as well as in avariety of therapeutic applications. The fluorescent agents can bind tothe enzyme carbonic anhydrase, which often is upregulated duringhypoxia.

Certain fluorophores, such as cyanine fluorophores with absorbance andemission spectra in the far-red and near-infrared regions, can havemolecular weights that far exceed the molecular weights of bindinggroups, such as carbonic anhydrase binding groups, and can have adominant effect on the physical, chemical, and biological properties ofan agent when linked to a binding group, which can negatively impact invitro and in vivo imaging performance. Chemical modification of thefluorophore moiety can overcome these limitations by allowingmanipulation of the physical, chemical and biological properties of theagent without directly modifying the binding group. Further, thechemical modifying moieties can influence binding affinity, bindingselectivity among multiple enzymes, and the nonspecific uptake of theagent in cells or tissues that are otherwise negative with respect tothe binding target. In particular, modification of a cyanine dye in sucha manner as to impart a significant net negative charge at physiologicalpH of, for example net charge of −3 to −6, can render a CA IX agentimpermeable to CA IX negative cells and favor rapid clearance from CA IXnegative tissues in vivo. In certain embodiments, the agents modified tohave net negative charge of −3 to −6 at neutral pH show unexpectedlyhigh performance with respect to binding affinity and target tobackground ratios in both in vitro and in vivo experiments.

In one aspect, the agents of the present invention comprise at least onecarbonic anhydrase binding moiety (CAB) chemically linked to afluorophore, wherein one or more chemical modifying moieties (M) arechemically linked to the fluorophore. Optionally, one or more linker (L)moieties can be used to chemically link the CAB to the fluorophore orthe M to the fluorophore. In some embodiments, the CAB is a moleculecontaining a sulfonamide and an aromatic or heteroaromatic moiety, andthe fluorophore is a cyanine dye.

A “carbonic anhydrase binding moiety” or CAB, as defined herein, is amolecule that specifically binds a carbonic anhydrase and/or reduces orprevents the catalytic activity of a carbonic anhydrase toward a naturalsubstrate for carbonic anhydrase, and/or antagonizes the binding of acarbonic anhydrase to its natural ligand. The binding between a CAB anda carbonic anhydrase may be covalent or non-covalent (for example,electrostatic interactions, hydrophobic interactions, van der Waalsinteractions, hydrogen bonding, dipole interactions, etc.). The bindingbetween a CAB and a carbonic anhydrase preferably is non-covalent.

In certain embodiments, the CAB has an affinity for a carbonicanhydrase, for example CA II, CA IV, CA IX, and CA XII.

The term “affinity” as used herein, refers to the ability of thecarbonic anhydrase agent to bind preferentially to and/or be retained bya carbonic anhydrase enzyme. Carbonic anhydrase agents with thischaracteristic are “targeted.” Affinity of a CAB or carbonic anhydraseagent can be measured, for example, by determining inhibition constantK_(i) towards a CA enzyme by measuring inhibition of activity of the CAenzyme for a substrate (e.g., CO₂) in the presence of the agent or, inthe case of membrane bound CAs such as CA IX, by measuring thedissociation constant K_(d) to live CA expressing cells by, for example,flow cytometry. In certain embodiments, the affinity of the carbonicanhydrase targeted agents described herein have a K_(i) or K_(d) to a CAof less than 100 nM. In certain preferred embodiments, the K_(i) orK_(d) of the agents described herein toward CA IX is less than 50 nM.

It is understood that the carbonic anhydrase targeting agents disclosedherein also include stereoisomeric forms thereof, or mixtures ofstereoisomeric forms thereof, or pharmaceutically acceptable salt formsthereof.

The “fluorophore” (F) may be any suitable chemical or substance which isused to provide fluorescent signal or contrast in imaging and that isdetectable by imaging techniques. In certain embodiments, F comprises,for example, a cyanine dye, carbocyanine dye, indocyanine dye, or apolymethine fluorescent dye.

In certain embodiments, F comprises a symmetrical cyanine dye. In otherembodiments, F comprises a non-symmetrical cyanine dye. In otherembodiments, F may also be modified with a plurality of chemicalmodifying moieties allowing optimization of the in vitro and in vivoproperties of the agent, and ultimately the performance of the agent asa fluorescence imaging agent.

As used herein, the term “chemically linked” is understood to meanconnected by an attractive force between atoms strong enough to allowthe combined aggregate to function as a unit. This includes, but is notlimited to, chemical bonds such as covalent bonds, non-covalent bondssuch as ionic bonds, metallic bonds, and bridge bonds, hydrophobicinteractions, hydrogen bonds, and van der Waals interactions.

As used herein, the term “functionality” is understood to mean areactive functional group that can be further modified or derivatizedwith another molecule. In one aspect, the reactive functional group isan amine, carboxylic acid, carboxylic ester, halogen, hydrazine,hydroxylamine, nitrile, isonitrile, isocyanate, isothiocyanate, thiol,maleimide, azide, alkyne, tetrazolyl, phosphonate, alkene, nitro, andnitroso.

As used herein, the term “chemical modifying group” or “M” is understoodto mean any moiety that can be used to alter the physical, chemical orbiological properties of the carbonic anhydrase targeting agent, suchas, without limitation, making the targeting agent more water soluble ormore dispersible in media for administration, increasing bindingspecificity, increasing or decreasing net molecular charge, decreasingimmunogenicity or toxicity, or modifying cell uptake, pharmacokinetic orbiodistribution profiles compared to the non-M modified carbonicanhydrase agents.

The “imaging reporter” or “IR” can be any suitable chemical or substancewhich is used to provide the contrast or signal in imaging and that isdetectable by imaging techniques. In certain preferred embodiments, theimaging reporter comprises one or more fluorophores, photoluminescentnanoparticles, radioisotopes, superparamagnetic agents, X-ray contrastagents, and ultrasound agents. It is understood that the IR can alsocomprise a therapeutic reporter such as a porphyrin used in photodynamictherapy and/or a radionuclide used in radiotherapy.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, and branched-chain alkylgroups. Moreover, the term “alkyl” (or “lower alkyl”) includes“substituted alkyls”, which refers to alkyl moieties having substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents may include, for example, a hydroxyl, a carbonyl (suchas a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CN and thelike. In certain embodiments, the alkyl is not substituted, i.e., it isunsubstituted. In instances where the term “alkyl” is used to describe achemical fragment that connects two portions of a compound, the term“alkyl” is understood to mean a diradical of a saturated aliphaticgroup, including a diradical of a straight-chain alkyl group, anddiradical of branched-chain alkyl group. To illustrate, the term alkylin “Br-alkyl-CO₂H” refers to an alkyl diradical that connects thebromine and carboxylic acid groups.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “heteroaryl” or“heteroaromatics.” The aromatic ring may be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls. In certain embodiments, the aryl is notsubstituted, i.e., it is unsubstituted. In instances where the term“aryl” is used to describe a chemical fragment that connects twoportions of a compound, the term “aryl” is understood to mean adiradical of an aromatic group. To illustrate, the term aryl in“Br-aryl-CO₂H” refers to an aryl diradical that connects the bromine andcarboxylic acid groups.

Terms “heterocyclic” and “heterocyclyl” are art recognized and refer toan aromatic or nonaromatic ring containing one or more heteroatoms. Theheteroatoms can be the same or different from each other. Examples ofheteratoms include, but are not limited to nitrogen, oxygen and sulfur.Aromatic and nonaromatic heterocyclic rings are well-known in the art.Some nonlimiting examples of aromatic heterocyclic rings includepyridine, pyrimidine, indole, purine, quinoline and isoquinoline.Nonlimiting examples of nonaromatic heterocyclic compounds includepiperidine, piperazine, morpholine, pyrrolidine and pyrazolidine.Examples of oxygen containing heterocyclic rings include, but notlimited to furan, oxirane, 2H-pyran, 4H-pyran, 2H-chromene, andbenzofuran. Examples of sulfur-containing heterocyclic rings include,but are not limited to, thiophene, benzothiophene, and parathiazine.Examples of nitrogen containing rings include, but not limited to,pyrrole, pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,imidazolidine, pyridine, piperidine, pyrazine, piperazine, pyrimidine,indole, purine, benzimidazole, quinoline, isoquinoline, triazole, andtriazine. Examples of heterocyclic rings containing two differentheteroatoms include, but are not limited to, phenothiazine, morpholine,parathiazine, oxazine, oxazole, thiazine, and thiazole. The heterocyclicring is optionally further substituted at one or more ring positionswith, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,carboxylic acid, —C(O)alkyl, —CO₂alkyl, carbonyl, carboxyl, alkylthio,sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester,heterocyclyl, aryl or heteroaryl moieties, —CF₃, —CN, or the like. Incertain embodiments, the heterocyclic or heterocyclyl group is notsubstituted, i.e., it is unsubstituted. In instances where the term“heterocyclyl” is used to describe a chemical fragment that connects twoportions of a compound, the term “heterocyclyl” is understood to mean adiradical of an aromatic or nonaromatic ring containing one or moreheteroatoms. To illustrate, the term heterocyclyl in“Br-heterocyclyl-CO₂H” refers to a heterocyclyl diradical that connectsthe bromine and carboxylic acid groups.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R⁵⁰, R⁵¹, R⁵² and R⁵³ each independently represent a hydrogen,an alkyl, an alkenyl, —(CH₂)_(m)—R⁶¹, or R⁵⁰ and R⁵¹, taken togetherwith the N atom to which they are attached complete a heterocycle havingfrom 4 to 8 atoms in the ring structure; R⁶¹ represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In certain embodiments, only oneof R⁵⁰ or R⁵¹ may be a carbonyl, e.g., R⁵⁰, R⁵¹ and the nitrogentogether do not form an imide. In other embodiments, R⁵⁰ and R⁵¹ (andoptionally R⁵²) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R⁶¹.

The term “oxo” is art-recognized and refers to a “═O” substituent. Forexample, a cyclopentane substituted with an oxo group is cyclopentanone.

The symbol “

” indicates a point of attachment.

Compounds described herein may contain charged functional groups, suchas a —SO₃ ⁻ group. It is understood that compounds containing suchcharged functional groups contain either (i) a sufficient number ofpositively charged functional group(s) and negatively charged functionalgroups so as to provide a charge-neutral compound, or (ii) one or morecounterions is present so that the compound is charge-neutral. If achemical structure is presented that would have an overall charge (asdepicted) it is understood that a sufficient number of counterion(s)(e.g., a halogen or acetate to counterbalance a positive charge, or ametal cation or ammonium group to counter-balance a negative charge) arepresent so as to provide a charge-neutral compound. Further, alltautomers of compounds depicted herein all within the scope of theinvention.

Throughout the description, where compositions and kits are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions andkits of the present invention that consist essentially of, or consistof, the recited components, and that there are processes and methodsaccording to the present invention that consist essentially of, orconsist of, the recited processing steps.

Various compounds are described by chemical structure and/or chemicalnames. In the event a discrepancy exists between a chemical structureand the chemical name provided for the structure, the chemical structurepredominates.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

In one aspect, the invention provides a carbonic anhydrase targetingagent comprising: a carbonic anhydrase targeting moiety comprising asulfonamide moiety optionally substituted with an aliphatic, aromatic orheteroaromatic moiety; and a fluorescent reporter chemically linked,optionally through a linker (L) moiety to the carbonic anhydrase moietywherein the fluorescent moiety bears a plurality of chemical modifyinggroups.

In another aspect, the invention provides a carbonic anhydrase targetingagent comprising: (a) a carbonic anhydrase targeting moiety comprising asulfonamide moiety optionally substituted with an aliphatic, aromatic orheteroaromatic moiety; and (b) an imaging reporter chemically linked,optionally through a linker (L) moiety to the carbonic anhydrase moiety.

In another aspect, the invention provides a carbonic anhydrase targetingagent represented by formula (I), or a salt thereof:

-   -   wherein:    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety;    -   Q is a substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl,        alkoxy, or thioalkyl group; or Q is absent;    -   L₁, L₂, and L₃ each represent independently for each occurrence        a bond or a linker moiety;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, C(CH₂Y₁)(CH₂Y₂), O, S,        or Se;    -   Y₁ and Y₂ are independently hydrogen or a C₁-C₂₀ aliphatic        group, each of which is optionally substituted with L₃-M;    -   M, independently for each occurrence, is hydrogen or a chemical        modifying moiety.

In another aspect, the invention provides a carbonic anhydrase targetingagent represented by formula (II), or a salt thereof:

-   -   wherein:    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety;    -   Q is a substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl,        alkoxy, or thioalkyl group; or Q is absent;    -   L₁, L₂, and L₃ each represent independently for each occurrence        a bond or a linker moiety;    -   R is, independently for each occurrence, hydrogen, substituted        or unsubstituted aryl, substituted or unsubstituted heteroaryl,        C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group, each        of which is optionally substituted with L₃-M;    -   n is 1, 2, or 3;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, C(CH₂Y₁)(CH₂Y₂), O, S,        or Se;

Y₁ and Y₂ are independently hydrogen or a C₁-C₂₀ aliphatic group, eachof which is optionally substituted with L₃-M; and

-   -   M, independently for each occurrence, is hydrogen or a chemical        modifying moiety.

In another aspect, the invention provides a carbonic anhydrase targetingagent of formula (III) or a salt thereof:

F-L-CAB,  (III)

-   -   wherein    -   F is a near infrared fluorochrome;    -   L is an optional linker; and    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety.

In another aspect, the invention provides a carbonic anhydrase targetingagent represented by formula (IV), or a salt thereof:

-   -   wherein:        -   Q is unsubstituted heteroaryl;        -   L₁ is one of the following:            -   (a) —C(O)N(R¹)-alkyl-C(O)N(R¹)-ω;            -   (b) —C(O)N(R¹)-alkyl-ω;            -   (c) —C(O)N(R¹)—[C₂₋₃ alkyl-O]_(z)—C₁₋₃alkyl-C(O)N(R¹)-ω;            -   (d) —C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω;            -   (e)                —C(O)N(R¹)-alkyl-C(O)N(R¹)—C₁₋₃alkyl-aryl-C(O)N(R¹)-ω;                or            -   (f) —C(O)N(R¹)— [C₂₋₃                alkyl-O]_(z)—C₁₋₃alkyl-C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω;            -   wherein z is an integer from about 3 to about 35, R¹                represents independently for each occurrence hydrogen or                alkyl, and ω is a bond to CAB;        -   L₂ is C₁₋₅alkyl;        -   M represents independently for each occurrence sulfonate or            —SO₃H; and        -   CAB is -aryl-SO₂NH₂, -heterocyclyl-SO₂NH₂,            -aryl-C(O)N(R′)-heteroaryl-SO₂NH₂,            -heteroaryl-C(O)N(R′)-heteroaryl-SO₂NH₂,            —C(O)N(R′)-heteroaryl-heteroaryl-SO₂NH₂, or            —C(O)N(R′)-aryl-heteroaryl-SO₂NH₂, wherein R′ represents            independently for each occurrence hydrogen or C₁₋₆ alkyl;            each aryl and heteroaryl are optionally substituted with 1            or 2 substituents independently selected from the group            consisting of alkyl and halogen, and each heterocyclyl is            optionally substituted with 1, 2, or 3 substituents            independently selected from the group consisting of alkyl,            halogen, amino, and oxo.

In another aspect, the invention provides a carbonic anhydrase targetingagent represented by formula (V), or a salt thereof:

-   -   wherein:    -   Q is C₁₋₆alkyl;    -   L₁ is one of the following:        -   (a) —C(O)N(R¹)-alkyl-C(O)N(R¹)-ω;        -   (b) —C(O)N(R¹)-alkyl-ω; or        -   (c) —C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω;            -   wherein R¹ represents independently for each occurrence                hydrogen or alkyl, and ω is a bond to CAB;    -   L₂ is C₁₋₅alkyl;    -   M represents independently for each occurrence sulfonate or        —SO₃H; and    -   CAB is -aryl-SO₂NH₂, -heterocyclyl-SO₂NH₂,        -aryl-C(O)N(R′)-heteroaryl-SO₂NH₂,        -heteroaryl-C(O)N(R′)-heteroaryl-SO₂NH₂,        —C(O)N(R′)-heteroaryl-heteroaryl-SO₂NH₂, or        —C(O)N(R′)-aryl-heteroaryl-SO₂NH₂, wherein R′ represents        independently for each occurrence hydrogen or C₁₋₆ alkyl; each        aryl and heteroaryl are optionally substituted with 1 or 2        substituents independently selected from the group consisting of        alkyl and halogen, and each heterocyclyl is optionally        substituted with 1, 2, or 3 substituents independently selected        from the group consisting of alkyl, halogen, amino, and oxo.

In another aspect, the invention provides a carbonic anhydrase targetingagent represented by formula (VI), or a salt thereof:

-   -   wherein:    -   CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety;    -   Q is a substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl,        alkoxy, or thioalkyl group; or Q is absent; L₁, L₂, and L₃ each        represent independently for each occurrence a bond or a linker        moiety;    -   R is, independently for each occurrence, hydrogen, substituted        or unsubstituted aryl, substituted or unsubstituted heteroaryl,        C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group, each        of which is optionally substituted with L₃-M;    -   n is 1, 2, or 3;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, C(CH₂Y₁)(CH₂Y₂), O, S,        or Se;    -   Y₁ and Y₂ are independently hydrogen or a C₁-C₂₀ aliphatic        group, each of which is optionally substituted with L₃-M;    -   M, independently for each occurrence, is hydrogen or a chemical        modifying moiety; and provided that there is at least one        occurrence of CAB.

In another aspect, the invention provides a compound of formula (A) or asalt thereof:

wherein CAB is a carbonic anhydrase targeting moiety comprising asulfonamide and an aliphatic, aromatic or heteroaromatic moiety;

-   -   L is, independently for each occurrence, a bond or a linker        moiety;    -   Q is selected from a group consisting of substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, and a thioalkyl group;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, selected from the group        consisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se;    -   Y₁ and Y₂ are independently selected from the group consisting        of H, and C₁-C₂₀ aliphatic group, each of which is optionally        substituted with L-M; and    -   M, independently for each occurrence, is a chemical modifying        moiety or is absent.

In another aspect, the invention provides a compound of formula (B), ora salt thereof:

-   -   wherein CAB is a carbonic anhydrase binding moiety comprising a        sulfonamide and an aliphatic, aromatic or heteroaromatic moiety;    -   L is, independently for each occurrence, a bond or a linker        moiety;    -   Q is selected from a group consisting of substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, and thioalkyl group, or    -   Q is absent;    -   R is, independently for each occurrence, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group, each        of which is optionally substituted with L-M, or R is absent;    -   n is 1, 2, or 3;    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X is, independently for each occurrence, selected from the group        consisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se;    -   Y₁ and Y₂ are independently selected from the group consisting        of H and C₁-C₂₀ aliphatic group, each of which is optionally        substituted with L-M; and    -   M, independently for each occurrence, is a chemical modifying        moiety or is absent.

Chemical Modifier M

The chemical modifier M can be an anionic moiety selected from the groupconsisting of a sulfonate, a carboxylate, phosphonate, phosphate, oriminodiacetate.

The chemical modifier M can be a hydrogen, alcohol, sulfonamide,sulfoxide, sulfone, ketone, an amino acid or polyamino acid, oligo- orpolyethylene glycol, an amine, a quaternary ammonium ion, or a sugarsuch as glucosamine, galactosamine or mannosamine.

The chemical modifier M can be a metal chelator, such as ethylenediaminetetraacetic acid, diethylenetriamine pentaacetic acid, andtetraazacyclododecane tetraacetic acid. In another aspect of theinvention, one or more metal chelating M groups are coordinated to ametal ion.

The chemical modifier M can be an ionizable group, for example, acarboxylate, sulfonate, phosphonate, phosphate or iminodiacetate, thatimpart a significant net negative charge on the agent. In certainembodiments, the net charge on the agent after chemical modification ofthe fluorophore with a plurality of ionizable modifying groups, isbetween −3 and −12 at neutral pH. In certain embodiments, the net chargeon the fluorophore moiety is −5.

In certain embodiments, the chemical modifier M comprises a biologicallyactive molecule, such as a drug or a radiotherapeutic moiety. In certainembodiments the biologically active molecule is linked to the agentthrough a linker that is cleavable through a biological or physicalmechanism including but not limited to enzymatic, thermal, acidcatalyzed or photochemical cleavage.

In certain embodiments, M represents independently for each occurrence asulfonate or —SO₃H. In certain embodiments, M represents independentlyfor each occurrence hydrogen, a sulfonate, or —SO₃H.

In certain embodiments M represents independently for each occurrence asubstituent selected from the group consisting of hydrogen, alcohol,sulfonic acid, sulfonate, polysulfonate, cysteic acid, sulfonamide,sulfoxide, sulfone, carboxylate, ketone, phosphonate, phosphate;iminodiacetate, ethylenediamine tetraacetic acid, diethylenetriaminepentaacetic acid, tetraazacyclododecane tetraacetic acid, an amino acidor polyamino acid, oligo- or polyethylene glycol, amine, quaternaryammonium ion, a sugar, glucosamine, galactosamine, mannosamine,polyethylene glycol (PEG), alkoxy polyethylene glycol, a branchedpolypropylene glycol, polypropylene glycol, a graft copolymer ofpoly-lysine and methoxypolyethyleneglycol, peptide, a lipid, a fattyacid, palmitate, phospholipid, a phospholipid-PEG conjugate, acarbohydrate, an iron oxide nanoparticle, naphthylalanine,phenylalanine, 3,3-diphenylpropylamine, taurine, a phosphonate, aphosphate, a carboxylate, and a polycarboxylate.

In certain embodiments, M represents independently for each occurrencehydrogen or —N(C₁₋₄ alkyl-SO₃H)₂.

In certain embodiments, the chemical modifying moiety M enhances thebinding selectivity of the carbonic anhydrase targeting agent forcarbonic anhydrase IX over other carbonic anhydrases including but notlimited to carbonic anhydrase II. In certain embodiments, the chemicalmodifying moiety M modifies the carbonic anhydrase targeting agent witha net negative charge ranging from −3 to −12 at neutral pH. In certainembodiments, the chemical modifying moiety M reduces the nonspecificcell membrane permeability of the carbonic anhydrase targeting agent. Incertain other embodiments, the chemical modifying moiety M reduces thenonspecific tissue accumulation of the carbonic anhydrase targetingagent when administered to a live animal.

Linker Moiety

In certain embodiments, the linker moiety, L, comprises a moietyselected from the group consisting of glycine, alanine, β-alanine,—NH—(CH₂)_(n)—C(═O)— where n=1-8, 4-aminomethylbenzoic acid, cysteicacid, glutamic acid, amino-polyethylene glycol-carboxylic acid,amino-polyethylene glycol amine, ethylenediamine, propylenediamine,spermidine, spermine, hexanediamine, and diamine-amino acids, such ashomolysine, lysine, ornithine, diaminobutyric acid and diaminopropionicacid, succinic acid, glutaric acid, suberic acid, and adipic acid. Incertain embodiments, L is a bond.

In certain other embodiments, each of L₁, L₂, and L₃ independentlycomprises —NH—(CH₂)_(n)—C(═O)— where n=1-8, or a diradical of a moietyselected from the group consisting of glycine, alanine, β-alanine,4-aminomethylbenzoic acid, cysteic acid, glutamic acid,amino-polyethylene glycol-carboxylic acid, amino-polyethylene glycolamine, ethylenediamine, propylenediamine, spermidine, spermine,hexanediamine, a diamine-amino acid, lysine, ornithine, diaminobutyricacid, diaminopropionic acid, succinic acid, glutaric acid, suberic acid,adipic acid, amide, triazole, urea, and thiourea.

In certain embodiments, L₁ is one of the following: (a)—C(O)N(R¹)-alkyl-C(O)N(R¹)-ω; (b) —C(O)N(R¹)-alkyl-ω; (c)—C(O)N(R¹)—[C₂₋₃ alkyl-O]_(z)—C₁₋₃ alkyl-C(O)N(R¹)-ω; (d)—C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω; (e) —C(O)N(R¹)-alkyl-C(O)N(R¹)—C₁₋₃alkyl-aryl-C(O)N(R¹)-ω; or (f) —C(O)N(R¹)—[C₂₋₃ alkyl-0]_(z)—C₁₋₃alkyl-C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω; wherein z is an integer fromabout 3 to about 35, R¹ represents independently for each occurrencehydrogen or alkyl, and ω is a bond to CAB.

In certain embodiments, L₂ is C₁₋₅alkyl. In certain embodiments, L₃ is abond.

In certain embodiments, L₂M is —(CH₂)₀₋₃CH₃.

Variable Q

In certain embodiments, Q can be selected from the group consisting of(i) a functionalized, substituted or unsubstituted aromatic orheteroaromatic ring, (ii) a functionalized, substituted or unsubstitutednitrogen containing heterocyclic ring, (iii) a functionalized,substituted or unsubstituted nitrogen containing 6-membered heterocyclicring, such as pyridine, pyrimidone, pyrazine, and pyridazine, (iv)functionalized, substituted or unsubstituted 6-membered aromatic ring,such as benzene, (v) a functionalized, substituted or unsubstitutedC₁-C₁₈alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group. In otherembodiments, Q is absent.

In certain embodiments, Q is unsubstituted heteroaryl, such aspyridinyl. In certain other embodiments, Q is alkyl, such as C₁₋₄ alkyl.

Exemplary Specific Carbonic Anhydrase Targeting Agents

The carbonic anhydrase targeting agent can be selected from the groupcomprising:

-   3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N-(4-oxo-4-((4-sulfamoylbenzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((4-sulfamoylbenzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-1-(4-sulfamoylphenyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-2-azaheptaheptacontan-77-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N-(4-oxo-4-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indol-3-ium-6-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-1-(4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-2-azaheptaheptacontan-77-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;-   3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate;    and-   3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1-sulfonate;    and pharmaceutically acceptable salts thereof.

In certain embodiments, the carbonic anhydrase targeting agent is one ofthe carbonic anhydrase targeting agents described in the Examples or apharmaceutically acceptable salt thereof.

The carbonic anhydrase targeting agents can be fluorescent in thefar-red or near-infrared spectral range.

In certain embodiments, the carbonic anhydrase targeting agent furthercomprises one or more chemical modifiers, independently, chemicallylinked to the CAB, L, and/or F or any combination thereof. Each of thefeatures of the carbonic anhydrase binding moieties, imaging reporters,linkers and biological modifiers are discussed in more detail below.

Carbonic Anhydrase Binding Moieties

Certain exemplary carbonic anhydrase binding moieties useful in thepractice of the invention are described in U.S. Pat. No. 7,833,737; andInternational Application Publication Nos. WO2006/137092, WO2008/124703,WO 2010/147666, and WO2010/065906, all of which are incorporated hereinby reference in their entirety.

In certain embodiments, the carbonic anhydrase binding (CAB) moiety is-aryl-SO₂NH₂, -heterocyclyl-SO₂NH₂, -aryl-C(O)N(R′)-heteroaryl-SO₂NH₂,-heteroaryl-C(O)N(R′)-heteroaryl-SO₂NH₂,—C(O)N(R′)-heteroaryl-heteroaryl-SO₂NH₂, or—C(O)N(R′)-aryl-heteroaryl-SO₂NH₂, wherein R′ represents independentlyfor each occurrence hydrogen or C₁₋₆ alkyl; each aryl and heteroaryl areoptionally substituted with 1 or 2 substituents independently selectedfrom the group consisting of alkyl and halogen, and each heterocyclyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of alkyl, halogen, amino, and oxo.

In certain embodiments, the CAB is -phenyl-SO₂NH₂; -pyridinyl-SO₂NH₂;1,3,4-thiadiazole-SO₂NH₂; -benzo[d]thiazole-SO₂NH₂;-phenyl-C(O)N(H)-pyridinyl-SO₂NH₂; -phenyl—C(O)N(H)-1,3,4-thiadiazole-SO₂NH₂;-pyridinyl-C(O)N(H)-1,3,4-thiadiazole-SO₂NH₂;—C(O)N(H)-pyridinyl-1,3,4-thiadiazole-SO₂NH₂; or—C(O)N(H)-phenyl-1,3,4-thiadiazole-SO₂NH₂; wherein each phenyl,pyridinyl, and thiadiazole are optionally substituted with 1 or 2substituents independently selected from the group consisting of alkyland halogen.

In certain embodiments, the carbonic anhydrase binding moiety is one ofthe following:

Imaging Reporters

It is understood that a variety of different imaging reporters, forexample, fluorescent and non-fluorescent reporters can be used toproduce a carbonic anhydrase targeting agent of the invention.

(a) Fluorescent Reporters

In certain embodiments the imaging reporter is a fluorophore molecule. A“fluorophore” includes, but is not limited to, a fluorochrome, afluorochrome quencher molecule, any organic or inorganic dye, metalchelate, or any fluorescent enzyme substrate, including proteaseactivatable enzyme substrates.

In certain embodiments, the carbonic anhydrase targeting agents comprisea fluorophore. In certain embodiments, the fluorophores are far red andnear infrared fluorochromes (NIRFs) with absorption and emission maximumbetween about 600 and about 1200 nm, more preferably between about 600nm and about 900 nm. It will be appreciated that the use offluorochromes with excitation and emission wavelengths in otherspectrums can also be employed in the compositions and methods of thepresent invention. Exemplary fluorochromes include but are not limitedto a carbocyanine fluorochrome and an indocyanine fluorochrome.

The near infrared fluorochromes preferably have an extinctioncoefficient of at least 50,000 M⁻¹cm⁻¹ per fluorochrome molecule inaqueous medium. The near infrared fluorochromes preferably also have (1)high quantum yield (i.e., quantum yield greater than 5% in aqueousmedium), (2) narrow excitation/emission spectrum, spectrally separatedabsorption and emission spectra (i.e., excitation and emission maximaseparated by at least 15 nm), (3) high chemical and photostability, (4)non-toxicity, (5) good biocompatibility, biodegradability andexcretability, and (6) commercial viability and scalable production forlarge quantities (i.e., gram and kilogram quantities) required for invivo and human use.

Certain carbocyanine or polymethine fluorescent dyes can be used toproduce the carbonic anhydrase targeting agents of the invention andinclude, for example, those described in U.S. Pat. Nos. 6,747,159;6,448,008; 6,136,612; 4,981,977; 5,268,486; 5,569,587; 5,569,766;5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536;6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; also WO 97/40104,WO 99/51702, WO 01/21624, and EP 1 065 250 A1; and Tetrahedron Letters41, 9185-88 (2000).

Various fluorochromes are commercially available and can be used toconstruct the carbonic anhydrase targeting agents of the invention.Exemplary fluorochromes include, for example, Cy5.5, Cy5 and Cy7 (GEHealthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, andAlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-5750(VisEn Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547,DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyteFluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor);and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and KodakX-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health).

Table 1 lists a number of exemplary fluorochromes useful in the practiceof the invention together with their spectral properties.

TABLE 1 Absorbance Fluorochrome ε_(max) M⁻¹cm⁻¹ max (nm) Cy5 250,000 649Cy5.5 250,000 675 Cy7 250,000 743 AlexaFlour660 132,000 663AlexaFlour680 184,000 679 AlexaFlour750 280,000 749 VivoTag680 (VT680)100,000 670 VivoTag-S680 220,000 674 VivoTag-S750 100,000 750 Dy677180,000 673 Dy682 140,000 690 Dy752 270,000 748 Dy780 170,000 782DyLight547 150,000 557 DyLight647 250,000 653 IRDye800CW 240,000 774IRDye800RS 200,000 767 IRDye700DX 165,000 689 ADS780WS 170,000 782ADS830WS 240,000 819 ADS832WS 190,000 824

In some embodiments, the fluorophore is substituted by a plurality ofchemical modifying groups. In one embodiment, the fluorophore isrepresented by formula VI:

-   -   or a salt thereof, wherein:    -   W represents a benzo-condensed, a naphtho-condensed or a        pyrido-condensed ring;    -   X, independently for each occurrence, is selected from the group        consisting of C(CH₂Y₁)(CH₂Y₂), O, S, and Se;    -   Y₁ and Y₂ are independently selected from the group consisting        of H and C₁-C₂₀ aliphatic group, each of which is optionally        substituted with L-M;    -   L, independently for each occurrence, represents a bond or a        linker moiety;    -   M, independently for each occurrence, represents a chemical        modifying moiety.

In one embodiment, the fluorochrome is represented by formula VII:

-   -   or a salt thereof, wherein:        -   X is independently selected from the group consisting of            C(CH₂Y₁)(CH₂Y₂), O, S, and Se;        -   Y₁ and Y₂ are independently selected from the group            consisting of H, C₁-C₂₀ aliphatic group and a C₁-C₂₀            aliphatic group substituted with —OR*, N(R*)₂ or —SR*;        -   W represents a benzo-condensed, a naphtho-condensed or a            pyrido-condensed ring;        -   R* is alkyl;        -   R₁ is selected from the group consisting of (CH₂)_(x)CH₃,            (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer            selected from 0 to 6 and n is an integer selected from 2 to            6;        -   R₄ is selected from the group consisting of (CH₂)_(x)CH₃,            (CH₂)_(n)SO₃ ⁻ and (CH₂)_(n)SO₃H, wherein x is an integer            selected from 0 to 6 and n is an integer selected from 2 to            6;        -   R₂ and R₃ are independently selected from the group            consisting of H, carboxylate, carboxylic acid, carboxylic            ester, amine, amide, sulfonamide, hydroxyl, alkoxyl, a            sulphonic acid moiety and a sulphonate moiety; and        -   Q is selected from a group consisting of a heteroaryl ring            substituted with a carboxyl group or 6-membered heteroaryl            ring substituted with a carbonyl group.

In certain embodiments of formula VII, Q is selected from a groupconsisting of (i) a carboxyl functionalized heterocyclic ring, (ii) acarboxyl functionalized nitrogen containing heterocyclic ring, (iii) acarboxyl functionalized nitrogen containing 6-membered heterocyclicring, such as pyridine, pyrimidone, pyrazine, and pyridazine, (iv) acarboxyl functionalized nitrogen containing 6-membered heterocyclicring, such as pyridine, (v) a carbonyl functionalized nitrogencontaining 6-membered heterocyclic ring, such as pyridine, and (vi) anisonicotinic acid, nicotinic acid and picolinic acid, and a groupselected from:

-   -   wherein, the carboxyl group is also in the form of an ester, an        activated ester or carbonyl halide capable of reacting with        nucleophiles, and can be, for example, a —C(O)O-benzotriazolyl,        —C(O)O—N-succinimidyl, —C(O)O-tetrafluorophenyl,        —C(O)O-pentafluorophenyl, —C(O)O-imidazole, or        —C(O)O-p-nitrophenyl.

In another embodiment, the fluorochrome is represented by formula VIII:

or a salt thereof, wherein:

-   -   X₁ and X₂ are independently selected from the group consisting        of C(CH₂K₁)(CH₂K₂), O, S and Se;    -   K₁ and K₂ are independently H or a C₁-C₂₀ aliphatic group; or K₁        and K₂ together are part of a substituted or unsubstituted        carbocyclic or heterocyclic ring;    -   Y₁ and Y₂ are each independently a benzo-condensed ring, a        naphtha-condensed ring or a pyrido-condensed ring;    -   n₁ is 1, 2, or 3;    -   R₂, R₁₁ and R₁₂ are independently H, halogen, C₁-C₆ alkyl, C₁-C₆        alkoxy, aryloxy, aryl, a sulfonate, an iminium ion, or any two        adjacent R₁₂ and R₁₁ substituents, when taken in combination,        form a 4-, 5-, or 6-membered carbocyclic ring optionally        substituted one or more times by C₁-C₆ alkyl or halogen;    -   R₁ and R₁₃ are (CH₂)_(x)CH₃, when x is an integer selected from        0 to 6; or R₁ and R₁₃ are independently (CH₂)_(n)SO₃ ⁻ or        (CH₂)_(n)SO₃H when n is an integer selected from 2 to 6;    -   R₃, R₄ and R₅ are independently selected from the group        consisting of H, carboxylate, carboxylic acid, carboxylic ester,        amine, amide, sulfonamide, hydroxyl, alkoxyl, a sulphonic acid        moiety and a sulphonate moiety;    -   R₆ is selected from the group consisting of an unsubstituted        C₁-C₂₀ aliphatic group, an unsubstituted aryl, or an        unsubstituted alkylaryl;    -   R₇ is selected from the group consisting of H, an unsubstituted        C₁-C₂₀ aliphatic group, an unsubstituted aryl, or an        unsubstituted alkylaryl, wherein R₇ is optionally substituted        with halogen; or    -   R₆ and R₇, taken together form a 4-, 5-, 6- or 7-membered        heterocyclic ring optionally substituted with halogen;    -   W is absent or is a group selected from the group consisting of        —SO₂NR₆—CHR₇—, —O—, —COO—, and —CONH—;    -   h=0-70; k=0 or 1; d=0-12; m=0-12; p=0-12.

Exemplary chemically modified fluorophores that can be used in thesynthesis of the carbonic anhydrase targeting agents of the inventioninclude, for example, those listed in Table 2.

TABLE 2 No. Fluorophore F1 

F2 

F3 

F4 

F5 

F6 

F7 

F8 

F9 

F10

F11

F12

  n = 3, 4, 8, 12, 24 F13

F14

F15

F16

F17

In certain embodiments, one or more fluorochrome molecules can bechemically linked to the carbonic anhydrase binding moiety to producethe fluorescent carbonic anhydrase targeting agents.

In the case where the imaging reporter is a fluorochrome molecule, theextinction coefficient of the carbonic anhydrase targeting agents can becalculated as the ratio of the absorbance of dye at its absorptionmaxima (for example at ˜670 nm for VivoTag 680) in a 1 cm path lengthcell to the concentration of particles using the formula ε=A/cl, where Ais absorbance, c is molar concentration and 1 is path length in cm.

It is understood that carbonic anhydrase targeting agent can be linkedto nanoparticles (for example, silicon containing nanoparticles) toproduce fluorescent or luminescent nanoparticles. Aggregates ofcrystalline silicon (as multiple or single crystals of silicon), poroussilicon, or amorphous silicon, or a combination of these forms, can formthe nanoparticle. Preferred fluorescent silicon nanoparticles have adiameter between about 0.5 nm to about 25 nm, more preferably betweenabout 2 nm and about 10 nm. The size of nanoparticles can be determinedby laser light scattering or by atomic force microscopy or othersuitable techniques.

Fluorescent silicon nanoparticles may have excitation and emissionspectra 200 to 2000 nm, however, preferred fluorescent siliconnanoparticles have excitation and emission maximum between about 400 nmand about 1200 nm (and preferably 500 nm-900 nm, for example, 500 nm-600nm, 600 nm-700 nm, 700 nm-800 nm, or 800 nm-900 nm). Preferredfluorescent silicon nanoparticles also have extinction coefficients ofat least 50,000 M⁻¹cm⁻¹ in aqueous medium. Although fluorescent siliconnanoparticles that have excitation and emission maximum between 400 nmand 1200 nm are preferred, it will be appreciated that the use offluorescent silicon nanoparticles with excitation and emissionwavelengths in other spectrums can also be employed in the compositionsand methods of the present invention. For example, in certainembodiments, the particles may have excitation approximately about300-350 nm, and emission approximately about 400-450 nm.

Fluorescent silicon nanoparticles may also have the followingproperties: (1) high quantum yield (i.e., quantum yield greater than 5%in aqueous medium), (2) narrow emission spectrum (i.e., less than 75 nm;more preferably less than 50 nm), (3) spectrally separated absorptionand emission spectra (i.e., separated by more than 20 nm; morepreferably by more than 50 nm), (3) have high chemical stability andphotostability (i.e., retain luminescent properties after exposure tolight), (4) are biocompatible (see below) or can be made morebiocompatible; (5) are non toxic or minimally toxic to cells or subjectsat doses used for imaging protocols, (as measured for example, by LD₅₀or irritation studies, or other similar methods known in the art) and/or(6) have commercial viability and scalable production for largequantities (i.e., gram and kilogram quantities) required for in vivo andhuman use.

Other exemplary fluorophores include metal oxide nanoparticles that arefluorescent and can be used in a variety of in vitro and vivoapplications. In one embodiment, the carbonic anhydrase binding moietiesare conjugated to fluorescent metal oxide nanoparticles with one or moreof the following features: (1) a polymer coating suitable for attachinga plurality of fluorochromes thereby achieving large extinctioncoefficients (in excess of 1,000,000 M⁻¹cm⁻¹), (2) a non-crosslinkedpolymer coating suitable for attaching from about 10 to about 300fluorochromes per particle, (3) a polymer coating suitable for attachinga plurality of fluorochromes in a manner that does not significantlycompromise the quantum yield of the fluorochromes (e.g., thenanoparticles retain at least 50% of the fluorescent signal that iscreated by substantially the same number of free fluorochromes whentested under the same conditions), and (4) a polymer coating that isamenable to efficient chemical linking of biomolecules with retention oftheir biological properties to yield molecular imaging agents. Thefluorescent metal oxide nanoparticles are highly stable molecularimaging agents in vitro, both before and after chemical linking offluorochromes and carbonic anhydrase targeting agents, but yet arelabile and/or degradable in vivo.

The carbonic anhydrase binding moiety can be linked to fluorescentquantum dots such as amine T2 MP EviTags (Evident Technologies) or QdotNanocrystals (Invitrogen). In general, fluorescent quantum dots arenanocrystals containing several atoms of a semiconductor material(including but not limited to those containing cadmium and selenium,sulfide, or tellurium; zinc sulfide, indium-antimony, lead selenide,gallium arsenide, and silica or ormosil, which have been coated withzinc sulfide to improve the properties of these fluorescent agents.

Furthermore, the carbonic anhydrase binding moiety can be conjugated tomolecules capable of eliciting photodynamic therapy. These include, butare not limited to, Photofrin, Lutrin, Antrin, aminolevulinic acid,hypericin, benzoporphyrin derivative, and select porphyrins.

In certain embodiments, one or more different fluorophore molecules canbe covalently linked to a cleavable (for example, an enzymaticallycleavable) oligopeptide, or alternatively, two substantially similarfluorophores can be covalently linked to the oligopeptide, atfluorescence-quenching permissive locations separated by a cleavagesite, for example, a proteolytic cleavage site, to produce the imagingagents of the invention.

In certain embodiments, a quencher is used to quench the fluorescentsignal from the fluorophore covalently linked to the oligopeptide. Forexample, an agent can be designed such that the quencher quenches thefluorescence of the fluorophore of the imaging agent when the agent isin an unactivated state, so that the imaging agent exhibits little or nosignal until it is activated. It is understood that the quencher can bea non-fluorescent agent, which when suitably located relative to afluorophore (i.e., at a fluorescence-quenching permissive location) iscapable of quenching the emission signal from the fluorophore. It isunderstood that certain of the foregoing fluorophores can act to quenchthe fluorescent signal of another spaced apart fluorophore, when the twofluorophores are positioned at fluorescence-quenching interactionpermissive locations.

A number of quenchers are available and known to those skilled in theart including, but not limited to4-{[4-(dimethylamino)-phenyl]azo}-benzoic acid (DABCYL), QSY®-7(9-[2-[(4-carboxy-1-piperidinyl)sulfonyl]phenyl]-3,6-bis(methylphenylamino)-xanthyliumchloride) (Molecular Probes, Inc., OR), QSY®-33 (Molecular Probes, Inc.,OR), ATTO612Q, ATTO580Q (ATTO-TEC, Germany); Black Hole Quenchers®(Bioresearch Technologies, Novato, Calif.), QXL™680 Acid (AnaSpec, SanJose Calif.), and fluorescence fluorophores such as Cy5 and Cy5.5 (e.g.,2-[5-[3-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benz[e]indol-2-ylidene]-1,3-pentadienyl]-3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benz[e]indolium,inner salt) (Schobel, Bioconjugate 10:1107, 1999). Other quenchingstrategies can be used, for example, using various solvents to quenchfluorescence of the agents.

Exemplary fluorophores that can quench the emission of otherfluorophores are represented in Table 3.

TABLE 3 No. Quencher Q1

Q2

In such embodiments, the two fluorophores or the fluorophore and thequencher are located within the intact imaging agent atfluorescent-quenching interaction permissive positions. In other words,a first fluorophore is located close enough in the intact imaging agentto a second fluophore (or quencher) to permit them to interactphotochemically with one another so that the second fluorophore (orquencher) quenches the signal from the first fluorophore. In the case ofthe imaging agents with two fluorophores, one fluorophore preferablyquenches the other fluorophore. For principles of quenching, see U.S.Pat. No. 6,592,847.

(b) Non-Fluorescent Reporters

The term “non-fluorescent reporter” as used herein, refers to a chemicalmoiety that is not fluorescent but which can be used to provide thecontrast or signal in imaging and is detectable by a non-fluorescentimaging technique. In certain embodiments, other non-fluorescentreporters can be chemically linked with the imaging agents, or can beadministered to a subject simultaneously or sequentially with theimaging agents of the invention. Such reporters can includephotoluminescent nanoparticles, radioisotopes, superparamagnetic agents,X-ray contrast agents, and ultrasound agents. A reporter may alsocomprise therapeutic reporters such as porphyrins, Photofrin®, Lutrin®,Antrin®, aminolevulinic acid, hypericin, benzoporphryrin derivativesused in photodynamic therapy, and radionuclides used for radiotherapy.

(i) Radioactive Reporters

The agents can include one or more radioactive labels. Radioisotopicforms of metals such as copper, gallium, indium, technetium, yttrium,and lutetium can be chemically linked to the metallic imaging agents andcan be used for nuclear imaging or therapeutic applications. Exemplaryradioactive labels include, without limitation, ^(99m)Tc, ¹¹¹In, ⁶⁴Cu,⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁷⁷Lu, and ⁶⁷Cu.

Other exemplary labels include, for example, ¹²³I, ¹²⁴I, ¹²⁵I, ¹¹C, ¹3N,¹⁵O, and ¹⁸F.

Other exemplary labels can be therapeutic radiopharmaceuticals includingfor example, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi,¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy,⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir.

Chelators or bonding moieties for diagnostic and therapeuticradiopharmaceuticals are also contemplated and can be chemicallyassociated with the imaging agents. Exemplary chelators can be selectedto form stable complexes with radioisotopes that have imageable gammaray or positron emissions, such as ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, and ⁶⁷Ga.Exemplary chelators include diaminedithiols,monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Chelatorsgenerally are tetradentate with donor atoms selected from nitrogen,oxygen and sulfur, and may include for example, cyclic and acyclicpolyaminocarboxylates such as diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), (DO3A),2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

(ii) Magnetic Reporters

Other exemplary reporters can include a chelating agent for magneticresonance agents. Such chelators can include for example,polyamine-polycarboxylate chelators or iminoacetic acid chelators thatcan be chemically linked to the agents.

Chelators for magnetic resonance imaging agents can be selected to formstable complexes with paramagnetic metal ions, such as Gd(III), Dy(III),Fe(III), and Mn(II), are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid, 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

In one embodiment, the carbonic anhydrase targeting agents areconjugated to superparamagnetic metal oxide nanoparticles that areeither (a) non-fluorescent or (b) are fluorescent and can be used in avariety of in vitro and vivo applications. Fluorescent metal oxidenanoparticles that also have magnetic properties can be used for MRI,thus providing a multi-modality imaging agent.

In certain embodiments, the imaging agents can include a fluorescentand/or non-fluorescent superparamagenetic metal oxide nanoparticle withone or more of the following features: (1) a polymer coating suitablefor attaching a plurality of agents (2) a non-crosslinked polymercoating suitable for attaching from about 10 to about 300 agents perparticle, and (3) a polymer coating that is amenable to efficientchemical linking of the agents with retention of their biologicalproperties to yield molecular imaging agents. The agent modified metaloxide nanoparticle can be a highly stable molecular imaging agent invitro, both before and after chemical linking of the agents, but yet arelabile and/or degradable in vivo.

The carbonic anhydrase targeting agent conjugated metal oxidenanoparticles can be formulated into a pharmaceutical compositionsuitable for administration to a subject, for example, an animal and/ora human subject.

(iii) Ultrasound Reporters

For ultrasound imaging, the imaging reporter can include gas-filledbubbles such as Levovist, Albunex, or Echovist, or particles or metalchelates where the metal ions have atomic numbers 21-29, 42, 44 or57-83. Examples of such compounds are described in Tyler et al.,Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, “EnhancementAgents for Ultrasound: Fundamentals,” Pharmaceuticals in MedicalImaging, pp. 682-87 (1990).

(iv) X-Ray Reporters

Exemplary reporters can comprise iodinated organic molecules or chelatesof heavy metal ions of atomic numbers 57 to 83. Examples of suchcompounds are described in M. Sovak, ed., “Radiocontrast Agents,”Springer-Verlag, pp. 23-125 (1984) and U.S. Pat. No. 4,647,447.

Linkers

Linker or spacer moieties (L) can be used to chemically link one or morechemical modifiers (M) to the fluorophore and/or to link the CAB moietyto Q or, if Q is absent, directly to the fluorophores of the agents ofthe present invention. Useful linker moieties include both natural andnon-natural amino acids and nucleic acids, peptides, such as glycine,β-alanine, γ-aminobutyric acid or aminocaproic acid, as well assynthetic linker molecules such as aminoethyl maleimide or aminomethylbenzoic acid, or a polymer such as homobifunctional orheterobifunctional polyethylene glycol (PEG). When the linker is apeptide, the peptide optionally may include proteolytic cleavage sitethat can be cleaved with a variety of agents, for example, an enzyme.

It is understood that there is no particular structural, size or contentlimitation for a given linker. Linkers can include, for example, avariety of functional groups such as maleimide, dithiopyridyl, thiol,azide, alkene, or alkyne that permit the assembly of molecules ofdiverse architecture.

Linkers can be homofunctional linkers or heterofunctional linkers. Forexample, amine (NH₂)-functionalized moieties can be reacted withbifunctional cross-linkers designed to react with amino groups.Particularly useful conjugation reagents that can facilitate formationof a linker or facilitate covalent linkage between, for example, afluorophore, and an enzymatically cleavable oligopeptide can include aN-hydroxysuccinimide (NHS) ester and/or a maleimide. The NHS ester canreact with the amine group of, for example, a peptide or fluorophore.The maleimide can react with the sulfhydryl group of another molecule.Other particularly useful linker moieties are bifunctional crosslinkerssuch as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), longchain-SPDP, maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS),succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC),succinimidyl iodoacetate (SIA).

In certain embodiments a linker, if present, may be a derivative of adiamine. A diamine moiety or derivative can provide a linker arm ofvarying lengths and chemistries for chemically linking molecules byderivatizing, optionally, with carboxylic acids. Non-limiting examplesof diamines include ethylenediamine (EDA), propylenediamine, spermidine,spermine, hexanediamine, and diamine-amino acids, such as homolysine,lysine, ornithine, diaminobutyric acid and diaminopropionic acid. Inother embodiments, moieties of an imaging agent can be chemically linkedto a dicarboxylic acid, for example, succinic acid, glutaric acid,suberic acid, or adipic acid. In one embodiment, the linker isaminoethylmaleimide.

In certain embodiments, a linker can be branched, for example glutamicacid or 5-(aminomethyl)isophthalic acid, or a dendrimer, such as alysine or glutamic acid dendrimer, with multiple M groups linked to asingle site on the fluorophore.

In certain embodiments, L is a functionalized, substituted orunsubstituted C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkylgroup. In other embodiments, L is functionalized, substituted orunsubstituted aromatic or heteroaromatic ring. In other embodiments, Lis absent.

In certain embodiments, a linker can be formed from an azide moiety thatcan react with substituted alkynes in an azide-acetylene Huisgen [3+2]cycloaddition. In certain embodiments the azide or alkyne linker canlink a polyethyleneglycol (PEG) moiety to, for example, an enzymaticallycleavable oligopeptide. Other contemplated linkers includepropargylglycine, pentanoyl, pentynoic acid, propargylic acid, and/orpropargylamine moieties.

In certain embodiments, the imaging reporters are directly chemicallylinked to the carbonic anhydrase binding moiety using reactive NHSesters groups on the IR which react with the amine group of theamino-functionalized CAB. In certain other embodiments, carboxylic acidgroups on the IR can be activated in situ by activating agents known inthe art, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate (HBTU),1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),N,N′-dicyclohexylcarbodiimide (DCC), N,N′-disuccinimidyl carbonate(DSC). In other embodiments, IRs containing a sulfhydryl or thiol group,can be chemically linked to the ITM via a bifunctional cross-linker thathas a second moiety that can react with a sulfhydryl (thiol) group. Suchcrosslinking agents include, for example and as described above, SPDP,long chain-SPDP, SIA, MBS, SMCC, and others that are well known in theart.

Useful linker moieties include both natural and non-natural amino acids,oligopeptides, for example, linear or cyclic oligopeptides, and nucleicacids. The linker can be a peptide or peptide moiety. The linker canoptionally include a proteolytic or non-proteolytic cleavage site, suchas an ester linkage, that can be cleaved due to pH changes at the siteof interest.

As used herein, the term “enzymatically cleavable oligopeptide” isunderstood to mean a peptide comprising two or more amino acids (asdefined herein) that are linked by means of a enzymatically cleavablepeptide bond. Also included are moieties that include a pseudopeptide orpeptidomimetic. Examples of cleavable peptide substrates can be found inU.S. Pat. No. 7,439,319.

The term “amino acid” as used herein is understood to mean an organiccompound containing both a basic amino group and an acidic carboxylgroup. Included within this term are natural amino acids (e.g., L-aminoacids), modified and unusual amino acids (e.g., D-amino acids), as wellas amino acids which are known to occur biologically in free or combinedform but usually do not occur in proteins. Natural amino acids include,but are not limited to, alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine,tyrosine, tryptophan, proline, and valine. Other amino acids include,but not limited to, arginosuccinic acid, citrulline, cysteine sulfinicacid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine, ornithine,carnitine, selenocysteine, selenomethionine, 3-monoiodotyrosine,3,5-diiodotryosine, 3,5,5′-triiodothyronine, and3,3′,5,5′-tetraiodothyronine.

Modified or unusual amino acids which can be used to practice theinvention include, but are not limited to, D-amino acids, hydroxylysine,dehydroalanine, pyrrolysine, 2-aminoisobutyric acid, gamma aminobutyricacid, 5-hydroxytryptophan, S-adenosyl methionine, S-adenosylhomocysteine, 4-hydroxyproline, an N-Cbz-protected amino acid,2,4-diaminobutyric acid, homoarginine, norleucine, N-methylaminobutyricacid, naphthylalanine, phenylglycine, .beta.-phenylproline,tert-leucine, 4-aminocyclohexylalanine, N-methyl-norleucine,3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine,4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid,trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid,1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoic acid.

As used herein, a “pseudopeptide” or “peptidomimetic” is a compoundwhich mimics the structure of an amino acid residue or a peptide, forexample, by using linking groups other than via amide linkages(pseudopeptide bonds) and/or by using non-amino acid substituents and/ora modified amino acid residue. A “pseudopeptide residue” means thatportion of a pseudopeptide or peptidomimetic that is present in apeptide. The term “pseudopeptide bonds” includes peptide bond isostereswhich may be used in place of or as substitutes for the normal amidelinkage. These substitute or amide “equivalent” linkages are formed fromcombinations of atoms not normally found in peptides or proteins whichmimic the spatial requirements of the amide bond and which shouldstabilize the molecule to enzymatic degradation. The followingconventional three-letter amino acid abbreviations are used herein:Ala=alanine; Aca=aminocaproic acid, Ahx=6-aminohexanoic acid,Arg=arginine; Asn=asparagines; Asp=aspartic acid; Cha=cyclohexylalanine;Cit=citrulline; Cys=cysteine; Dap=diaminopropionic acid; Gln=glutamine;Glu=glutamic acid; Gly=glycine; His=histidine; Ile=isoleucine;Leu=leucine; Lys=lysine; Met=methionine; Nal=naphthylalanine;Nle=norleucine; Orn=ornithine; Phe=phenylalanine; Phg=phenylglycine;Pro=praline; Sar=sarcosine; Ser=serine; Thi=Thienylalanine;Thr=threonine; Trp=tryptophan; Tyr=tyrosine; and Val=valine. Use of theprefix D- indicates the D-isomer of that amino acid; for exampleD-lysine is represented as D-Lys.

The peptides can be synthesized using either solution phase chemistry orsolid phase chemistry or a combination of both (Albericio, Curr.Opinion. Cell Biol., 8, 211-221 (2004), M. Bodansky, Peptide Chemistry:A Practical Textbook, Springer-Verlag; N. L. Benoiton, Chemistry ofPeptide Synthesis, 2005, CRC Press).

Selective or orthogonal amine protecting groups may be required toprepare the agents of the invention. As used herein, the term “amineprotecting group” means any group known in the art of organic synthesisfor the protection of amine groups. Such amine protecting groups includethose listed in Greene, “Protective Groups in Organic Synthesis” JohnWiley & Sons, New York (1981) and “The Peptides: Analysis, Synthesis,Biology, Vol. 3, Academic Press, New York (1981). Any amine protectinggroup known in the art can be used. Examples of amine protecting groupsinclude, but are not limited to, the following: 1) acyl types such asformyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromaticcarbamate types such as benzyloxycarbonyl (Cbz or Z) and substitutedbenzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxycarbonyl, and9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate types suchas tert-butyloxycarbonyl (Boc), ethoxycarbonyl,diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkylcarbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl;5) alkyl types such as triphenylmethyl and benzyl; 6) trialkylsilanesuch as trimethylsilane; and 7) thiol containing types such asphenylthiocarbonyl and dithiasuccinoyl. Also included in the term “amineprotecting group” are acyl groups such as azidobenzoyl,p-benzoylbenzoyl, o-benzylbenzoyl, p-acetylbenzoyl, dansyl,glycyl-p-benzoylbenzoyl, phenylbenzoyl, m-benzoylbenzoyl,benzoylbenzoyl.

In certain embodiments the enzymatically cleavable oligopeptide caninclude oligo-L-arginine, oligo-L-lysine, oligo-L-aspartic acid oroligo-L-glutamic acid.

The enzymatically cleavable oligopeptide is cleavable by at least oneenzyme chosen from hydrolases, elastases, cathepsins, matrixmetalloproteases, peptidases, exopeptidases, endopeptidases,carboxypeptidases, glycosidases, lipases, nucleases, lyases, amylases,phospholipases, phosphatases, phosphodiesterases, sulfatases, serineproteases, subtilisin, chymotrypsin, trypsin, threonine proteases,cysteine proteases, calpains, papains, caspases, aspartic acidproteases, pepsins, chymosins, glutamic acid proteases, renin,reductases, and parasitic, viral and bacterial enzymes.

Chemical Modifiers

Depending upon the intended use, the carbonic anhydrase targeting agentscan comprise one or more chemical modifiers (M), which can alter thephysical, chemical or biological properties of the carbonic anhydrasetargeting agent. In particular, a plurality of Ms can be chemicallylinked to the phluorophore moiety of the agent. The Ms can be the sameor can be different for each occurrence. For example, the Ms may renderthe carbonic anhydrase agents more useful for biological imaging, thatis, for example, more water soluble, or more dispersible in media foradministration, with increased binding specificity, or less immunogenic,or less toxic, or with reduced non-specific binding, alteredbiodistribution and pharmacokinetic compared to an unsubstituted orlesser substituted fluorophore moiety.

For example, incorporation of methoxypolyethylene glycol (mPEG) orpolypeptides or a plurality of anionic Ms may function to modify thepharmacodynamics and blood clearance rates of the carbonic anhydraseagents in vivo. Other Ms can be chosen to accelerate the clearance ofthe carbonic anhydrase targeting agents from background tissue, such asmuscle or liver, and/or from the blood, thereby reducing the backgroundinterference and improving image quality. Additionally, the Ms can beused to favor a particular route of excretion, e.g., via the kidneysrather than via the liver. The Ms can also aid in formulating probes inpharmaceutical compositions or may be used to alter or preserve thesignal reporting properties of the carbonic anhydrase targeting agents.In particular, chemical linking of polyethylene glycol (PEG) or aderivative thereof to carbonic anhydrase targeting agents can result inlonger blood residence time (longer circulation) and decreasingimmunogenicity.

Exemplary modifiers include polyethylene glycol (PEG) and derivativesthereof (for example, alkoxy polyethylene glycol (for example,methoxypolyethylene glycol, ethoxypolyethylene glycol and the like),branched polypropylene glycol, polypropylene glycol, a graft copolymerof poly-lysine and methoxypolyethyleneglycol, amino acids, peptides,lipids, fatty acids, palmitate, phospholipids, phospholipid-PEGconjugates, carbohydrates (such as dextran, amino-dextran,carboxymethyl-dextran), iron oxide nanoparticles, sulfonates,polysulfonates, cysteic acid, naphthylalanine, phenylalanine, and3,3-diphenylpropylamine taurine, phosphonates, phosphates, carboxylatesand polycarboxylates.

In certain embodiments, the chemical modifier M is an anionic moietyselected from the group consisting of carboxylate, phosphonate,phosphate, iminodiacetate, cysteic acid, or taurine.

In certain embodiments, the chemical modifier M is a sulfonate orpolysulfonate.

In certain embodiments, the chemical modifier M is a hydrogen, alcohol,sulfonamide, sulfoxide, sulfone, ketone, an amino acid such as glutamicacid or taurine, a polyamino acid such as polycysteic acid, oligo- orpolyethylene glycol, an amine, a quaternary ammonium ion, or acarbohydrate such as glucosamine, galactosamine or mannosamine.

In certain embodiments, the chemical modifier M is a metal chelator,such as ethylenediamine tetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or tetraazacyclododecane tetraacetic acid(DOTA). In another aspect of the invention, one or more metal chelatingM groups are coordinated to a metal ion.

In certain embodiments, as discussed above, the biological modifier maybe a PEG moiety that has a molecular weight, for example, from about 0.1kDa to about 50 kDa, about 5 kDa to about 35 kDa, or about 10 kDa toabout 30 kDa. Alternatively, the PEG may be dPEG, functionalized at adiscrete molecular weight, for example, of about 1100 daltons.

In certain embodiments, the PEG ismethoxyPEG₍₅₀₀₀₎-succinimidylpropionate (mPEG-SPA),methoxyPEG₍₅₀₀₀₎-succinimidylsuccinate (mPEG-SS). Such PEGS arecommercially available from Nektar Therapeutics or SunBiowest orLaysanBio or NOF.

The PEG moiety can be conjugated to reactive amines on the carbonicanhydrase targeting agent via a carboxyl functionality. Alternatively,the PEG modifier can be conjugated to the carbonic anhydrase targetingagent by using a thiol reactive cross linker and then reacting with athiol group on the PEG.

In one embodiment, the PEG may be branched, or Y-shaped, as availablefrom JenKem USA or NOF, or comb-shaped, or synthesized by coupling twoor more PEGs to a small molecule such as glutamic acid.

The omega position of PEG may include a hydroxyl group or a methoxygroup and the PEG may also contain an amino group in the omega position.Such an amino group can in turn be coupled to a variety of agents. Inanother embodiment of the present invention, the biological modifier canbe a pegylated poly-L-lysine or a pegylated poly-D-lysine.

In other embodiments, the biological modifier can bepolyvinylpyrrolidone (PVP)-type polymers. The biological modifier can bea functionalized polyvinylpyrrolidone, for example, carboxy or aminefunctionalized on one (or both) ends of the polymer (as available fromPolymersource) or within the polymer chain.

Alternatively, the biological modifier can include PolyN-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine,carboxy, etc.), Poly(N-isopropyl acrylamide) or functionalizedpoly(N-isopropylacrylamide).

Biological modifiers can include straight or branched chain acyl groups,such as pentynoyl; acidic groups, such as succinyl; lower alkyl groups,such as methyl, ethyl, propyl, etc.; carboxyalkyl groups, such ascarboxyethyl; haloalkyl groups, such as trifluoromethyl; and the like.

In general, the chemical linking of Ms does not adversely affect theaffinity and/or binding properties of the carbonic anhydrase targetingagents.

Exemplary Carbonic Anhydrase Targeting Agents, and Formulations

Useful carbonic anhydrase targeting agents can be created using one ormore of the carbonic anhydrase binding moieties, imaging reporters,biological modifiers, and linkers described hereinabove using standardchemistries known in the art. Depending upon the particular application,the carbonic anhydrase targeting agents should be water soluble or waterdispersible (i.e., sufficiently soluble or suspendable in aqueous orphysiological media solutions). In one embodiment, the carbonicanhydrase targeting agent is water soluble or dispersible in aqueousmedia, and is biocompatible, i.e., non-toxic having, for example, anLD₅₀ of greater than about 50 mg/kg body weight. The carbonic anhydrasetargeting agents preferably do not have any undesired phototoxicproperties and/or display low serum protein binding affinity.

A list of exemplary carbonic anhydrase targeting agents is provided inTable 4.

TABLE 4 Com- pound No. Structure Name IV-1 

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3- oxo-3-((4-sulfamoylbenzyl)amino)propyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)- 1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1- sulfonate IV-2 

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3- oxo-3-(((5-sulfamoylpyrimidin-2-yl)methyl)amino)propyl)carbamoyl) pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol- 3-ium-3-yl)propane-1-sulfonateIV-3 

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-(((2-sulfamoylbenzo[d]thiazol-5- yl)methyl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol- 3-ium-3-yl)propane-1-sulfonateIV-4 

2-((1E,3Z,5E)-5-(6,8- bis(bis(carboxymethyl)carbamoyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol- 2(3H)-ylidene)-3-(5-(4-sulfamoylbenzylcarbamoyl)pyridin-2- yl)penta-1,3-dienyl)-6,8-bis(bis(carboxymethyl)carbamoyl)-3- ethyl-1,1-dimethyl-1H-benzo[e]indolium IV-5 

3-(2-((1E,3Z,5E)-5-(6,8-bis(1-carboxy-2-sulfoethylcarbamoyl)-1,1-dimethyl-3- (3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-(3-oxo-3-(5- sulfamoyl-1,3,4-thiadiazol-2-ylamino)propylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-6,8-bis(1-carboxy-2-sulfoethylcarbamoyl)-1,1-dimethyl- 1H-benzo[e]indolium-3-yl)propane-1-sulfonate IV-6 

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-7 

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-(3- ((E)-3-methyl-5-sulfamoyl-1,3,4-thiadiazol-2(3H)-ylideneamino)-3- oxopropylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-1,1-dimethyl-6,8- disulfo-1H-benzo[e]indolium-3-yl)propane-1-sulfonate IV-8 

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-(((5-((5-sulfamoyl-1,3,4-thiadiazol-2- yl)carbamoyl)pyridin-2-yl)methyl)carbamoyl)pyridin-2- yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-9 

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-(4- sulfamoylbenzylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-3,3-dimethyl-5- sulfo-3H-indolium-1-yl)propane-1-sulfonate IV-10

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-(3- sulfamoylbenzylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-3,3-dimethyl-5- sulfo-3H-indolium-1-yl)propane-1-sulfonate IV-11

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-(3-oxo-3-(3- sulfamoylbenzylamino) propylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-3,3- dimethyl-5-sulfo-3H-indolium-1-yl)propane-1-sulfonate IV-12

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((5- sulfamoylpyrimidin-2-yl)methylcarbamoyl)pyridin-2-yl) penta-1,3-dienyl)-3,3-dimethyl-5-sulfo-3H- indolium-1-yl)propane-1-sulfonateIV-13

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-(3-oxo-3-(5-sulfamoyl- 1,3,4-thiadiazol-2-ylamino)propylcarbamoyl)pyridin-2- yl)penta-1,3-dienyl)-3,3-dimethyl-5-sulfo-3H-indolium-1-yl)propane-1- sulfonate IV-14

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-(3-((E)-3-methyl-5- sulfamoyl-1,3,4-thiadiazol-2(3H)-ylideneamino)-3- oxopropylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-3,3-dimethyl-5- sulfo-3H-indolium-1-yl)propane-1-sulfonate IV-15

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((5-(5-sulfamoyl-1,3,4-thiadiazol-2-ylcarbamoyl)pyridin-2- yl)methylcarbamoyl)pyridin-2-yl)penta- 1,3-dienyl)-3,3-dimethyl-5-sulfo-3H-indolium-1-yl)propane-1-sulfonate IV-16

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((6-oxo-6-(((5-((5- sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)pyridin-2- yl)methyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1- yl)propane-1-sulfonate IV-17

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((6-((3-((3-methyl-5- sulfamoyl-1,3,4-thiadiazol-3-ium-2-yl)amino)-3-oxopropyl)amino)-6- oxohexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3- dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1- sulfonate IV-18

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((6-oxo-6-((3-oxo-3-((5- sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)amino)hexyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1- yl)propane-1-sulfonate IV-19

2-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(2-sulfoethyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)ethanesulfonate IV-20

2-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(2-sulfoethyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((3-oxo-3-((5-sulfamoyl-1,3,4- thiadiazol-2-yl)amino)propyl)amino)hexyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1- dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)ethanesulfonate IV-21

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((3-oxo-3-((5-sulfamoyl-1,3,4- thiadiazol-2-yl)amino)propyl)amino)hexyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol- 3-ium-3-yl)propane-1-sulfonateIV-22

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-23

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-(((5-((3-oxo-3-((5-sulfamoyl- 1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2- yl)methyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol- 3-ium-3-yl)propane-1-sulfonateIV-24

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6- sulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-(((5-((3-oxo-3-((5-sulfamoyl- 1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2- yl)methyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6-sulfo-1H-benzo[e]indol-3- ium-3-yl)propane-1-sulfonate IV-25

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6- sulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((3-oxo-3-((5-sulfamoyl-1,3,4- thiadiazol-2-yl)amino)propyl)amino)hexyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6-sulfo-1H-benzo[e]indol-3- ium-3-yl)propane-1-sulfonate IV-26

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((3-((3-oxo-3-((5-sulfamoyl- 1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl) benzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane- 1-sulfonate IV-27

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((15-oxo-15-((5-sulfamoyl-1,3,4-thiadiazol- 2-yl)amino)-3,6,9,12-tetraoxapentadecyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-28

3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol- 2(3H)-ylidene)-3-(5-(3-oxo-3-(5-sulfamoyl-1,3,4-thiadiazol-2- ylamino)propylcarbamoyl)pyridin-2-yl)penta-1,3-dienyl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indolium-6-sulfonate IV-29

3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-(5-sulfamoyl-1,3,4-thiadiazol-2-ylcarbamoyl)pyridin-2- yl)penta-1,3-dienyl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indolium-6-sulfonate IV-30

3-(2-((1E,3Z,5E)-5-(3-ethyl-1,1- dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((5-sulfamoyl- 1,3,4-thiadiazol-2-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1- sulfonate IV-31

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)- ylidene)-3-(5-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium-3-yl)propane- 1-sulfonate IV-32

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-5,6,8- trisulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((5- sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-5,6,8-trisulfo-1H-benzo[e]indol-3-ium-3-yl)propane- 1-sulfonate IV-33

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4- ((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-34

3-(5-((1-carboxy-2- sulfoethyl)carbamoyl)-2-((1E,3Z,5E)-5-(5-((1-carboxy-2- sulfoethyl)carbamoyl)-3,3-dimethyl-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5- ((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2- yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl- 3H-indol-1-ium-1-yl)propane-1-sulfonate IV-35

3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol- 2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8- sulfo-1H-benzo[e]indol-3-ium-6-sulfonate IV-36

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(4-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-37

  N = 3, 4, 8, 12, or 24 N/A IV-38

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3- oxo-3-((4-sulfamoylbenzyl)amino)propyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)- 1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1- sulfonate IV-39

3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N- (4-oxo-4-((4-sulfamoylbenzyl)amino)butyl) sulfamoyl)- 1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate IV-40

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((4- sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1- sulfonate IV-41

3-(2-((1E,3E,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3- dimethyl-5-(N-methyl-N-(4-oxo-4-((4-sulfamoylbenzyl)amino)butyl) sulfamoyl)- 3H-indol-1-ium-1-yl)propane-1-sulfonate IV-42

2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8- sulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-3-ium-6-sulfonate IV-43

3-(2-((1E,3E,5E,7E)-7-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-5-(N-methyl-N-(4-oxo-4-((4- sulfamoylbenzyl)amino)butyl)sulfamoyl)- 3H-indol-1-ium-1-yl)propane-1- sulfonate IV-44

3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((5-oxo-5-((4- sulfamoylbenzyl)amino)pentyl)carbamoyl) pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indol- 3-ium-6-sulfonate IV-45

3-(2-((1E,3E,5E,7E)-7-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-5-(N-methyl-N-(4-oxo-4-((5- sulfamoyl-1,3,4-thiadiazol-2-yl)amino)butyl)sulfamoyl)-3H-indol-1- ium-1-yl)propane-1-sulfonate IV-46

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6- oxo-6-((4-((5-sulfamoyl-1,3,4-thiadiazol-2- yl)carbamoyl)benzyl)amino)hexyl) carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)- 1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1- sulfonate IV-47

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-48

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-oxo-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)butyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-49

3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N- (4-oxo-4-((4-((5-sulfamoyl-1,3,4-thiadiazol-2- yl)carbamoyl)benzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-50

  N = 3, 4, 8, 12, or 24 N/A IV-51

  N = 3, 4, 8, 12, or 24 N/A IV-52

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-53

3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-sulfamoylpyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indol-3-ium-6- sulfonate IV-54

3-(2-((1E,3Z,5Z)-5-(6,8-disulfo-3-(3- sulfopropyl)naphtho[2,1-d]thiazol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5- sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2- yl)penta-1,3-dien-1-yl)-6,8-disulfonaphtho[2,1-d]thiazol-3-ium-3- yl)propane-1-sulfonate IV-55

3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl- 1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1- sulfonate IV-56

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-(5-oxo-5-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)pentyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1- sulfonate IV-57

  n = 0-6 N/A IV-58

  n = 0-6 N/A IV-59

3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8- disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(4-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3- yl)propane-1-sulfonate IV-60

3-(2-((1E,3E,5E)-5-(3,3-dimethyl-5- sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3- dimethyl-5-(N-methyl-N-(4-oxo-4-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2- yl)amino)propyl)amino)butyl)sulfamoyl)- 3H-indol-1-ium-1-yl)propane-1- sulfonate

The symbol “N/A” indicates that a chemical name was not available.

The imaging agents disclosed herein can be formulated into apharmaceutical composition suitable for administration to a subject, forexample, an animal and/or a human. The pharmaceutical composition caninclude one or more imaging agents and one or more excipients, forexample, a stabilizer in a physiologically relevant carrier.

For in vivo use, the compositions of the present invention can beprovided in a formulation suitable for administration to a subject, forexample, an animal or a human. Accordingly, the formulations include theagents together with a physiologically relevant carrier suitable for thedesired form and/or dose of administration. The term, “physiologicallyrelevant carrier” is understood to mean a carrier in which the agentsare dispersed, dissolved, suspended, admixed and physiologicallytolerable, i.e., can be administered to, in, or on the subject's bodywithout undue discomfort, or irritation, or toxicity. The preferredcarrier is a fluid, preferably a liquid, more preferably an aqueoussolution; however, carriers for solid formulations, topicalformulations, inhaled formulations, ophthalmic formulations, andtransdermal formulations are also contemplated as within the scope ofthe invention.

It is contemplated that the agents can be administered orally orparenterally. For parenteral administration, the agents can beadministered intravenously, intramuscularly, cutaneously,percutaneously, subcutaneously, rectally, nasally, vaginally, andocularly. Thus, the composition may be in the form of, e.g., solidtablets, capsules, pills, powders including lyophilized powders,colloidal suspensions, microspheres, liposomes granulates, suspensions,emulsions, solutions, gels, including hydrogels, pastes, ointments,creams, plasters, irrigation solutions, drenches, osmotic deliverydevices, suppositories, enemas, injectables, implants, sprays, oraerosols. The pharmaceutical compositions can be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, 20th edition, 2000, ed. A. R. Germaro,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

It is understood that the formulation of the agents, the choice of modeof administration, the dosages of agents administered to the subject,and the timing between administration of the agents and imaging iswithin the level of skill in the art.

Applications

It is understood that carbonic anhydrase targeting agents can be used ina variety of imaging and therapeutic applications.

(a) Imaging Methods

The present invention provides methods for in vitro and in vivo imagingusing the imaging agents disclosed herein. For a review of opticalimaging techniques, see, e.g., Alfano et al., Ann. NY Acad. Sci.820:248-270 (1997); Weissleder, Nature Biotechnology 19, 316-317 (2001);Ntziachristos et al., Eur. Radiol. 13:195-208 (2003); Graves et al.,Curr. Mol. Med. 4:419-430 (2004); Citrin et al., Expert Rev. AnticancerTher. 4:857-864 (2004); Ntziachristos, Ann. Rev. Biomed. Eng. 8:1-33(2006); Koo et al., Cell Oncol. 28:127-139 (2006); and Rao et al., Curr.Opin. Biotechnol. 18:17-25 (2007).

Optical imaging includes all methods from direct visualization withoutuse of any device and use of devices such as various scopes, cathetersand optical imaging equipment, for example computer based hardware fortomographic presentations. The imaging agents are useful with opticalimaging modalities and measurement techniques including, but not limitedto: endoscopy; fluorescence endoscopy; luminescence imaging; timeresolved transmittance imaging; transmittance imaging; nonlinearmicroscopy; confocal imaging; acousto-optical imaging; photoacousticimaging; reflectance spectroscopy; spectroscopy; coherenceinterferometry; interferometry; optical coherence tomography; diffuseoptical tomography and fluorescence mediated molecular tomography(continuous wave, time domain frequency domain systems and earlyphoton), and measurement of light scattering, absorption, polarization,luminescence, fluorescence lifetime, quantum yield, and quenching.

An imaging system useful in the practice of the invention typicallyincludes three basic components: (1) an appropriate light source forinducing excitation of the imaging agent, (2) a system for separating ordistinguishing emissions from light used for fluorophore excitation, and(3) a detection system. The detection system can be hand-held orincorporated into other useful imaging devices, such as intraoperativemicroscopes. Exemplary detection systems include an endoscope, catheter,tomographic system, hand-held imaging system, or an intraoperativemicroscope.

Preferably, the light source provides monochromatic (or substantiallymonochromatic) light. The light source can be a suitably filtered whitelight, i.e., bandpass light from a broadband source. For example, lightfrom a 150-watt halogen lamp can be passed through a suitable bandpassfilter commercially available from Omega Optical (Brattleboro, Vt.).Depending upon the system, the light source can be a laser. See, e.g.,Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994;Ntziachristos et al., Proc. Natl. Acad. Sci. USA 97:2767-2772, 2000; andAlexander, J. Clin. Laser Med. Surg. 9:416-418, 1991. Information onlasers for imaging can be found, for example, at Imaging DiagnosticSystems, Inc., Plantation, Fla. and various other sources. A high passor bandpass filter can be used to separate optical emissions fromexcitation light. A suitable high pass or bandpass filter iscommercially available from Omega Optical, Burlington, Vt.

In general, the light detection system can be viewed as including alight gathering/image forming component and a light/signaldetection/image recording component. Although the light detection systemcan be a single integrated device that incorporates both components, thelight gathering/image forming component and light detection/imagerecording component are discussed separately.

A particularly useful light gathering/image forming component is anendoscope. Endoscopic devices and techniques which have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. Photochem. Photobiol. B 52:131-135, 1999),ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132, 1997), colonand rectum (Mycek et al., Gastrointest. Endosc. 48:390-394, 1998; andStepp et al., Endoscopy 30:379-386, 1998), bile ducts (Izuishi et al.,Hepatogastroenterology 46:804-807, 1999), stomach (Abe et al., Endoscopy32:281-286, 2000), bladder (Kriegmair et al., Urol. Int. 63:27-31, 1999;and Riedl et al., J. Endourol. 13:755-759, 1999), lung (Hirsch et al.,Clin Cancer Res 7:5-220, 2001), brain (Ward, J. Laser Appl. 10:224-228,1998), esophagus, and head and neck regions can be employed in thepractice of the present invention.

Other types of light gathering components are catheter-based devices,including fiber optics devices. Such devices are particularly suitablefor intravascular imaging. See, e.g., Tearney et al., Science276:2037-2039, 1997; and Circulation 94:3013, 1996.

Still other imaging technologies, including phased array technology(Boas et al., Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994; Chance,Ann. NY Acad. Sci. 838:29-45, 1998), optical tomography (Cheng et al.,Optics Express 3:118-123, 1998; and Siegel et al., Optics Express4:287-298, 1999), intravital microscopy (Dellian et al., Br. J. Cancer82:1513-1518, 2000; Monsky et al., Cancer Res. 59:4129-4135, 1999; andFukumura et al., Cell 94:715-725, 1998), confocal imaging (Korlach etal., Proc. Natl. Acad. Sci. USA 96:8461-8466, 1999; Rajadhyaksha et al.,J. Invest. Dermatol. 104:946-952, 1995; and Gonzalez et al., J. Med.30:337-356, 1999) and fluorescence molecular tomography (FMT)(Nziachristos et al., Nature Medicine 8:757-760, 2002; U.S. Pat. No.6,615,063, PCT WO 03/102558, and PCT WO 03/079015) can be used with theimaging agents of the invention. Similarly, the imaging agents can beused in a variety of imaging systems, for example, (1) the IVIS® ImagingSystems: 100 Series, 200 Series (Xenogen, Alameda, Calif.), (2) SPECTRUMand LUMINA (Xenogen, Alameda, Calif.), (3) the SoftScan® or the eXploreOptix™ (GE Healthcare, United Kingdom), (4) Maestro™ and Nuance™-2Systems (CRi, Woburn, Mass.), (5) Image Station In-Vivo FX fromCarestream Molecular Imaging, Rochester, N.Y. (formerly Kodak MolecularImaging Systems), (6) OV100, IV100 (Olympus Corporation, Japan), (7)Cellvizio Mauna Kea Technologies, France), (8)] NanoSPECT/CT or HiSPECT(Bioscan, Washington, D.C.), (9) CTLM® or LILA™ (Imaging DiagnosticSystems, Plantation, Fla.), (10) DYNOT™ (NIRx Medical Technologies, GlenHead, N.Y.), and (11) NightOWL Imaging Systems by Berthold Technologies,Germany.

A variety of light detection/image recording components, e.g., chargecoupled device (CCD) systems or photographic film, can be used in suchsystems. The choice of light detection/image recording depends onfactors including the type of light gathering/image forming componentbeing used. It is understood, however, that the selection of suitablecomponents, assembling them into an optical imaging system, andoperating the system is within ordinary skill in the art.

For agents that have magnetic properties, MRI imaging well known in theart can also be applied in the practice of the invention. For a reviewof MRI techniques see Westbrook, Handbook of MRI Technique, 2^(nd)Edition, 1999, Blackwell Science. It is possible that images obtained,for example, by optical imaging and by magnetic resonance imaging can beco-registered or fused with one another to provide additionalinformation about the item being imaged. Furthermore, multi-modalityimaging systems (i.e., combined optical and MR imaging systems) can beused to create combined optical MR images.

In addition, the compositions and methods of the present invention canbe used for other imaging compositions and methods. For example, theagents of the present invention can be imaged by other imagingmodalities, such as, X-ray, computed tomography (CT), MR imaging,ultrasound, positron emission tomography (PET), and single photoncomputerized tomography (SPECT).

In addition, the compositions and methods of the present invention canbe used in combination with other imaging compositions and methods. Forexample, the agents of the present invention can be imaged by opticalimaging protocols either alone or in combination with other traditionalimaging modalities, such as, X-ray, computed tomography (CT), MRimaging, ultrasound, positron emission tomography (PET), and singlephoton computerized tomography (SPECT). For instance, the compositionsand methods of the present invention can be used in combination with CTor MRI to obtain both anatomical and molecular informationsimultaneously, for example, by co-registration of with an imagegenerated by another imaging modality. The compositions and methods ofthe present invention can also be used in combination with X-ray, CT,PET, ultrasound, SPECT and other optical and MR contrast agents oralternatively, the agents of the present invention may also includeimaging agents, such as iodine, gadolinium atoms and radioactiveisotopes, which can be detected using CT, PET, SPECT, and MR imagingmodalities in combination with optical imaging. The imaging agents canbe linked to or incorporated in the agents.

(i) In Vivo Imaging Methods

With respect to optical in vivo imaging, such a method comprises (a)administering to a subject one or more of the carbonic anhydrasetargeting agents described herein, (b) allowing sufficient time topermit the agent to distribute with the subject, and (c) detecting asignal emitted by the carbonic anhydrase targeting agent. The signalemitted by the agent can be used to construct an image, for example, atomographic image. The foregoing steps can be repeated at predeterminedtime intervals thereby to permit evaluation of the emitted signals ofthe carbonic anhydrase targeting agents in the subject over time.

In another in vivo imaging method, the method comprises (a)administering to a subject one or more of the carbonic anhydrasetargeting agents described herein that contains a fluorochrome; (b)allowing sufficient time to permit the carbonic anhydrase targetingagent to distribute within the subject; (c) exposing the subject tolight of a wavelength absorbable by the fluorochrome, and (d) detectinga signal emitted by the carbonic anhydrase targeting agent. Theforegoing steps can be repeated at predetermined time intervals therebyto permit evaluation of the emitted signals of the carbonic anhydrasetargeting agents in the subject over time. The illuminating and/ordetecting steps (steps (c) and (d), respectively) can be performed usingan endoscope, catheter, tomographic system, planar system, hand-heldimaging system, goggles, or an intraoperative microscope.

Before or during these steps, a detection system can be positionedaround or in the vicinity of a subject (for example, an animal or ahuman) to detect signals emitted from the subject. The emitted signalscan be processed to construct an image, for example, a tomographicimage. In addition, the processed signals can be displayed as imageseither alone or as fused (combined) images.

In addition, it is possible to practice an in vivo imaging method thatselectively detects and images one, two or more molecular imagingprobes, including the carbonic anhydrase targeting agentssimultaneously. In such an approach, for example, in step (a) notedabove, two or more imaging probes whose signal properties aredistinguishable from one another are administered to the subject, eitherat the same time or sequentially, wherein at least one of the molecularimaging probes is a carbonic anhydrase agent. The use of multiple probespermits the recording of multiple biological processes, functions ortargets.

The subject may be a vertebrate, for example, a mammal, for example, ahuman. The subject may also be a non-vertebrate (for example, C.elegans, drosophila, or another model research organism, etc.) used inlaboratory research.

Information provided by such in vivo imaging approaches, for example,the presence, absence, or level of emitted signal can be used to detectand/or monitor a disease in the subject. Exemplary diseases include,without limitation, autoimmune disease, bone disease, cancer,cardiovascular disease, environmental disease, dermatological disease,immunologic disease, inherited disease, infectious disease, metabolicdisease, neurodegenerative disease, ophthalmic disease, and respiratorydisease. In addition, in vivo imaging can be used to assess the effectof a compound or therapy by using the imaging agents, wherein thesubject is imaged prior to and after treatment with the compound ortherapy, and the corresponding signal/images are compared.

The carbonic anhydrase targeting agents also can be used in in vivoimaging method where cells labeled with the carbonic anhydrase targetingagent are administered to the recipient. The cells can be labeled withthe carbonic anhydrase targeting agents either in vivo or ex vivo. Inthe ex vivo approach, cells can be derived directly from a subject orfrom another source (e.g., from another subject, cell culture, etc.).The carbonic anhydrase targeting agents can be mixed with the cells toeffectively label the cells and the resulting labeled cells administeredto the subject into a subject in step (a). Steps (b)-(d) then arefollowed as described above. This method can be used for monitoringtrafficking and localization of certain cell types, including T-cells,tumor cells, immune cells and stem cells, and other cell types. Inparticular, this method may be used to monitor cell-based therapies.

It is understood that the formulation of the carbonic anhydrasetargeting agents, the choice of mode of administration, the dosages ofcarbonic anhydrase targeting agents administered to the subject, and thetiming between administration of the carbonic anhydrase targeting agentsand imaging is within the level of skill in the art.

The foregoing methods can be used to determine a number of indicia,including tracking the localization of the carbonic anhydrase targetingagent in the subject over time or assessing changes or alterations inthe metabolism and/or excretion of the carbonic anhydrase targetingagent in the subject over time. The methods can also be used to followtherapy for such diseases by imaging molecular events and biologicalpathways modulated by such therapy, including but not limited todetermining efficacy, optimal timing, optimal dosing levels (includingfor individual patients or test subjects), and synergistic effects ofcombinations of therapy.

The methods and compositions of the invention can be used to help aphysician or surgeon to identify and characterize areas of disease, suchas cancers and specifically hypoxic tumors, or other hypoxic tissues, todistinguish diseased and/or hypoxic tissues from normal tissues, such asdetecting specific regions of hypoxia within a tumor or other tissuesthat are difficult to detect using ordinary imaging techniques, and tofurther assess said tissues as candidates for particular treatmentregimens, or gauge the prognosis such as likelihood of metastasis.

The methods and compositions of the invention can also be used in thedetection, characterization and/or determination of the localization ofa disease, including early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state.

The methods and compositions of the invention can also be used tomonitor and/or guide various therapeutic interventions, such as surgicalprocedures, and monitoring drug therapy, including cell based therapies.The methods described herein can also be used to assess therapeuticefficacy of various treatment regimens, including but not limited tothose designed to reduce tumor acidosis and metastasis or variousradiotherapeutics. The methods of the invention can also be used inprognosis of a disease or disease condition.

With respect to each of the foregoing, examples of such disease ordisease conditions that can be detected or monitored (before, during orafter therapy) include inflammation (for example, inflammation caused byarthritis, for example, rheumatoid arthritis), cancer (for example,colorectal, ovarian, lung, breast, prostate, cervical, testicular, skin,brain, gastrointestinal, pancreatic, liver, kidney, bladder, stomach,leukemia, mouth, esophageal, and bone), cardiovascular disease (forexample, atherosclerosis and inflammatory conditions of blood vessels,ischemia, stroke, thrombosis, and disseminated intravascularcoagulation), dermatologic disease (for example, Kaposi's Sarcoma,psoriasis, and allergic dermatitis), ophthalmic disease (for example,macular degeneration, and diabetic retinopathy), infectious disease (forexample, bacterial, viral, fungal and parasitic infections, includingAcquired Immunodeficiency Syndrome, Malaria, Chagas Disease, andSchistosomiasis), immunologic disease (for example, an autoimmunedisorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetesmellitus, lupus erythematosis, myasthenia gravis, and Graves disease),central nervous system disease (for example, a neurodegenerativedisease, such as Parkinson's disease or Alzheimer's disease,Huntington's Disease, amyotrophic lateral sclerosis, and prion disease),inherited diseases, metabolic diseases, environmental diseases (forexample, lead, mercury and radioactive poisoning, and skin cancer),bone-related disease (for example, osteoporosis, primary and metastaticbone tumors, and osteoarthritis), neurodegenerative disease, andsurgery-related complications (such as graft rejection, organ rejection,alterations in wound healing, fibrosis or other complications related tosurgical implants).

The methods and compositions described herein can, therefore, be used,for example, to detect and/or quantify the presence and/or localizationof carbonic anhydrase in a subject, including humans, for instance intumor cells, and to detect and/or quantify the presence and/orlocalization of hypoxic tissue, including the presence of hypoxicregions within a tumor. The methods and compositions described hereincan also be used to detect and/or quantify carbonic anhydrase IXassociated with diseases, disorders and conditions, including but notlimited to preneoplastic/neoplastic disease including areas at risk foracute occlusion (i.e., vulnerable plaques) in coronary and peripheralarteries, regions of expanding aneurysms, unstable plaque in carotidarteries, and ischemic areas. The methods and compositions of theinvention can also be used in identification and evaluation of celldeath, injury, apoptosis, necrosis, and hypoxia. The methods andcompositions can also be used for drug delivery and to monitor drugdelivery, especially when drugs or drug-like molecules are chemicallyattached to the fluorescent probes. Exemplary drug molecules includechemotherapeutic and cytostatic agents and photodynamic agents includingbut not limited to Photofrin, Lutrin, Antrin, aminolevulinic acid,hypericin, benzoporphyrin derivative, and porphyrins.

In addition, the methods and compositions described herein can be usedto image hypoxia (oxygen deficiency) in a subject. The method comprisesadministering to a subject (for example, a human or animal) an amount ofone or more of the carbonic targeting agents described herein sufficientto facilitate hypoxia imaging. After sufficient time to permit the agentto distribute within the animal or distribute within the area to beimaged, the presence and/or amount of the agent is determined. Thepresence and/or amount of the agent can then be used to create an image,for example, a tomographic image, representative of oxygen depletionwithin the tissues of the subject.

(ii) In Vitro Imaging Methods

With respect to in vitro imaging, the imaging agents can be used in avariety of in vitro assays. For example, an exemplary in vitro imagingmethod comprises: (a) contacting a sample, for example, a biologicalsample, with one or more of the carbonic anhydrase targeting agentsdescribed herein; (b) allowing the agent(s) to interact with abiological target in the sample; (c) optionally, removing unbound agent;and (d) detecting a signal emitted from the agent thereby to determinewhether the agent has been activated by or bound to the biologicaltarget. When the carbonic anhydrase targeting agent comprises afluorochrome, step (d) further comprises illuminating the sample withlight of a wavelength absorbable by the fluorochrome to produce theemitted signal.

After an agent has been designed, synthesized, and optionallyformulated, it can be tested in vitro by one skilled in the art toassess its biological and performance characteristics. For instance,different types of cells grown in culture can be used to assess thebiological and performance characteristics of the agent. Cellularuptake, binding or cellular localization of the agent can be assessedusing techniques known in the art, including, for example, fluorescentmicroscopy, FACS analysis, immunohistochemistry, immunoprecipitation, insitu hybridization and Forster resonance energy transfer (FRET) orfluorescence resonance energy transfer. By way of example, the agentscan be contacted with a sample for a period of time and then washed toremove any free agents. The sample can then be viewed using anappropriate detection device such as a fluorescent microscope equippedwith appropriate filters matched to the optical properties of afluorescent agent. Fluorescence microscopy of cells in culture orscintillation counting is also a convenient means for determiningwhether uptake and binding has occurred. Tissues, tissue sections andother types of samples such as cytospin samples can also be used in asimilar manner to assess the biological and performance characteristicsof the agents. Other detection methods including, but not limited toflow cytometry, immunoassays, hybridization assays, and microarrayanalysis can also be used.

(b) Therapeutic Applications

Certain of the carbonic anhydrase targeting agents described herein, forexample, agents containing a radiolabel and drug molecule, can be usedto ameliorate a symptom of, or treat, a particular disease or disorder.The method comprises (a) administering an amount of one or more theagents described herein sufficient to impart a therapeutic effect in thesubject; and (b) permitting sufficient time for the agent to distributewithin the subject or otherwise localize in a region of the subject tobe treated and then, (c) depending on the therapeutic agent, optionallyactivating the agent to impart a therapeutic effect. For example, whenthe therapeutic agent is a radiolabel, no subsequent activation isrequired. However, when the therapeutic agent is a photoreactive agent,for example, a dye used in photodynamic therapy, the agent may beactivated by exposing the agent to light having a wavelength thatactivates the agent. As a result, the agents can be used to treat acondition of interest, for example, a cancer, immune disorder,inflammatory disorder, vascular disorder and the like. Furthermore theagents can be used to inhibit acidosis in a hypoxic tumor, or otherregion of interest in the subject, or reduce tumor cell proliferationfrom hypoxic regions.

The invention will now be illustrated by means of the followingexamples, which are given for the purpose of illustration only andwithout any intention to limit the scope of the present invention.

Examples

The compounds of the present invention can be synthesized from readilyavailable starting materials following standard methods and procedures.The following non limiting examples demonstrate the synthesis ofexemplary fluorescent carbonic anhydrase agents. Representativematerials and methods that may be used in preparing the materials of theinvention are described further below. Unless otherwise stated, allchemicals and solvents (reagent grade) are used as commercially obtainedwithout further purification. Synthesized compounds are characterized byHPLC on a 2695 system using either a Phenomenex Phenyl-hexyl column (3μ,100×4.6 mm) or C18 column (5μ, 250×4.6 mm) with a flow rate of 0.5 to1.0 mL/min. A linear gradient of A (25 mM ammonium formate, +5%methanol) and B (acetonitrile) was used. Typically, the gradient startedat 0-20% of B and changed to 25-90% of B over 10 to 20 min.Chromatograms were monitored using a 2998 photodiode array detector andZQ2000 MS mass detector. Preparative HPLC was performed on a Variansystem using a Phenomenex C18 column (250×21 mm) at 20 mL/min usingsimilar gradient as the analytical run.

Example 1: Synthesis of exemplary compound3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N-(4-oxo-4-((4-sulfamoylbenzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[B1]

4-Aminomethylbenzenesulfonamide (2.7 mg, 12 μmol) and the succinimidylester of fluorochrome F1 (10 μmol) and 5.5 μL N-methylmorpholine (50μmol) were combined in 130 μL of anhydrous DMF in a sealed vial androtated at 25° C. for 1 hour. The crude product was precipitated byaddition of 1.5 mL of ethyl acetate, centrifuged, decanted and driedunder vacuum. The product was purified by HPLC and the mass confirmed byLCMS, calculated for [M+H]⁺ 1206.2, found 1206.2.

Example 2: Synthesis of exemplary compound3-ethyl-2-((1E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indol-3-ium-6-sulfonate[B6]

4-Aminomethylbenzenesulfonamide (3.2 mg, 14 μmol), the succinimidylester of fluorochrome F6 (12 μmol), 6.5 μL N-methylmorpholine (60 μmol),dimethylaminopropyl ethyl carbodiimide (2.0 mg, 10.5 μmol), andhydroxybenzotriazole (2.0 mg, 15 μmol) were combined in 150 μL ofanhydrous DMF in a sealed vial and rotated at 25° C. for 3 hours. Thecrude product was precipitated by addition of 1.5 mL of ethyl acetate,centrifuged, decanted and dried under vacuum. The product was purifiedby HPLC and the mass confirmed by LCMS, calculated for [M+H]⁺, 1120.3,found 1120.3.

Example 3: Synthesis of exemplary compound2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-3-ium-6-sulfonate[B5]

4-aminomethylbenzenesulfonamide (3.5 mg, 16 μmol), the succinimidylester of fluorochrome F5 (13 μmol) and 7.2 μL N-methylmorpholine (65μmol), were combined in 110 μL of anhydrous DMF in a sealed vial androtated at 25° C. for 1 hour. The crude product was precipitated byaddition of 1.5 mL of ethyl acetate, centrifuged, decanted and driedunder vacuum. The product was purified by HPLC and the mass confirmed byLCMS, calculated for [M+H]+ 1308.1, found 1308.3.

Example 4: Synthesis of exemplary compound3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((4-sulfamoylbenzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[B3]

4-Aminomethylbenzenesulfonamide (3.0 mg, 13 μmol), the succinimidylester of fluorochrome F3 (11 μmol) and 6.2 μL N-methylmorpholine (55μmol), were combined in 105 μL of anhydrous DMF in a sealed vial androtated at 25° C. for 1 hour. The crude product was precipitated byaddition of 1.5 mL of ethyl acetate, centrifuged, decanted and driedunder vacuum. The product was purified by HPLC and the mass confirmed byLCMS, calculated for [M+2H]²⁺711.1, found 711.5.

Example 5: Synthesis of exemplary compound3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-1-(4-sulfamoylphenyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-2-azaheptaheptacontan-77-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[B12]

4-Aminomethylbenzenesulfonamide (3.0 mg, 13 μmol), the succinimidylester of fluorochrome F12 (n=24) (11 μmol) and 6.2 μL N-methylmorpholine(55 μmol), were combined in 105 μL of anhydrous DMF in a sealed vial androtated at 25° C. for 1 hour. The crude product was precipitated byaddition of 1.5 mL of ethyl acetate, centrifuged, decanted and driedunder vacuum. The product was purified by HPLC and the mass confirmed byLCMS, calculated for [M+2H+3NH₃]²⁺1244.4, found 1244.5.

Example 6: Synthesis of exemplary compound2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-sulfamoylbenzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-3-ium-6-sulfonate[C5]

4-aminoethylbenzenesulfonamide (3.7 mg, 16 μmol), the succinimidyl esterof fluorochrome F5 (13 μmol) and 7.2 μL N-methylmorpholine (65 μmol),are combined in 110 μL of anhydrous DMF in a sealed vial and rotated at25° C. for 1 hour. The crude product is precipitated by addition of 1.5mL of ethyl acetate, centrifuged, decanted and dried under vacuum. Theproduct is purified by HPLC and the mass confirmed by LCMS.

Example 7: Synthesis of Intermediate Compound4-(aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide [AO]

Part A: Acetazolamide (1 g, 4.5 μmol) was dissolved 10 mL of methanoland 1.5 mL of concentrated hydrochloric acid and refluxed for 4 h. Thesolution was cooled to 0° C. in and neutralized by addition of 0.5 mL of1 M HEPES buffer, pH 7.0 followed by 1 M NaOH until a pH of 7. Methanolwas removed under vacuum then cooled to 4° C. and pure5-amino-1,3,4-thiadiazole-2-sulfonamide was allowed to crystalize out ofsolution.

Part B: 5-Amino-1,3,4-thiadiazole-2-sulfonamide, (100 mg, 560μmol)N-BOC-4-aminomethyl benzoic acid (140 mg, 560 μmol),dimethylaminopropyl ethyl carbodiimide (117 mg, 610 μmol) andhydroxybenzotriazole (84 mg, 610 μmol) were combined in 600 μL ofanhydrous DMF in a sealed reaction tube and rotated at room temperaturefor 48 h. The crude solution was diluted with 1 mL of water resulting inprecipitation of the product followed by slow addition of 1 M NaOH untila pH of 8.5 was achieved (˜1.5 mL) resulting in a clear solution. Thesolution was purified by prep HPLC and the purified product deprotectedin 600 μL of 95% TFA/water for 15 minutes then dried under vacuum toyield 150 mg (85%) of4-(aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide AO.

Example 8: Synthesis of exemplary compound3-(2-((1E,3E,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6-(N-methyl-N-(4-oxo-4-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)amino)butyl)sulfamoyl)-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[A1]

4-(Aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (5 mg, 16μmol), the succinimidyl ester of fluorochrome F1 (16 μmol) andN-methylmorpholine (10 μL) were combined in 100 μL of anhydrous DMF in asealed vial and rotated at 37° C. for 1 h. The crude product wasprecipitated by addition of 1.5 mL of ethyl acetate, centrifuged,decanted and dried under vacuum. The product was purified by HPLC andthe mass confirmed by LCMS, calculated for [M+H]+ 1333.2; found 1333.1.

Example 9: Synthesis of exemplary compound3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[A5]

4-(Aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (5 mg, 16μmol), the succinimidyl ester of fluorochrome F5 (16 μmol) andN-methylmorpholine (10 μL) were combined in 100 μL of anhydrous DMF in asealed vial and rotated at 37° C. for 1 h. The crude product wasprecipitated by addition of 1.5 mL of ethyl acetate, centrifuged,decanted and dried under vacuum. The product was purified by HPLC andthe mass confirmed by LCMS, calculated for [M+H]+ 1435.1; found 1435.1.

Example 10: Synthesis of exemplary compound3-ethyl-2-01E,3Z,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-8-sulfo-1H-benzo[e]indol-3-ium-6-sulfonate[A6]

4-(Aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (5 mg, 16μmol), the succinimidyl ester of fluorochrome F6 (16 μmol) andN-methylmorpholine (10 μL) were combined in 100 μL of anhydrous DMF in asealed vial and rotated at 37° C. for 1 h. The crude product wasprecipitated by addition of 1.5 mL of ethyl acetate, centrifuged,decanted and dried under vacuum. The product was purified by HPLC andthe mass confirmed by LCMS, calculated for [M+H]+ 1247.2; found 1247.3.

Example 11: Synthesis of exemplary compound3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((6-oxo-6-((4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)benzyl)amino)hexyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[A3]

4-(Aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (5 mg, 16μmol), the succinimidyl ester of fluorochrome F3 (16 μmol) andN-methylmorpholine (10 μL) were combined in 100 μL of anhydrous DMF in asealed vial and rotated at 37° C. for 1 h. The crude product wasprecipitated by addition of 1.5 mL of ethyl acetate, centrifuged,decanted and dried under vacuum. The product was purified by HPLC andthe mass confirmed by LCMS, calculated for [M+2H]²+774.6; found 775.0.

Example 12: Synthesis of exemplary compound3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-1-(4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)phenyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-2-azaheptaheptacontan-77-yl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[A12]

4-(Aminomethyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide (5 mg, 16μmol), the succinimidyl ester of fluorochrome F12 (n=24) (16 μmol) andN-methylmorpholine (10 μL) were combined in 100 μL of anhydrous DMF in asealed vial and rotated at 37° C. for 1 h. The crude product wasprecipitated by addition of 1.5 mL of ethyl acetate, centrifuged,decanted and dried under vacuum. The product was purified by HPLC andthe mass confirmed by LCMS, [M+3H]³⁺ calculated 854.9, found 855.4.

Example 13: Synthesis of Intermediate Compound3-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)propanamide [CO]

Part A: Acetazolamide (1 g, 4.5 μmol) was dissolved 10 mL of methanoland 1.5 mL of concentrated hydrochloric acid and refluxed for 4 h. Thesolution was cooled to 0° C. in and neutralized by addition of 0.5 mL of1 M HEPES buffer, pH 7.0 followed by 1 M NaOH until a pH of 7. Methanolwas removed under vacuum then cooled to 4° C. and pure5-amino-1,3,4-thiadiazole-2-sulfonamide was allowed to crystallize outof solution.

Part B: 5-amino-1,3,4-thiadiazole-2-sulfonamide (20 mg, 110 μmol)N-FMOCβ-alanine (31 mg, 100 μmol), dimethylaminopropyl ethyl carbodiimide(EDC) (21 mg, 110 μmol) and hydroxybenzotriazole (HOBT) (15 mg, 110μmol) were combined in 200 μL of anhydrous DMF in a sealed reaction tubeand rotated at room temperature for 48 h. The crude product was purifiedby prep HPLC, then deprotected by dissolving in 125 μL of 20%diethylamine in DMF for 10 min followed by precipitation with ether togive 3-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)propanamide as a whitesolid.

Example 14: Synthesis of Exemplary Compound3-(2-((1E,3Z,5E)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[C5]

3-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)propanamide (5 mg, 20 μmol)was combined with the succinimidyl ester of fluorochrome F5 (15 μmol)and 10 μL of N-methylmorpholine in 200 μL of anhydrous DMF in a sealedreaction tube and rotated at 37° C. for 2 h. The crude product wasprecipitated by addition of 1.5 mL of ethyl acetate, then purified byprep HPLC and the mass confirmed by LCMS, calculated for [M+H]+ 1373.1,found 1373.2.

Example 15: Synthesis of Exemplary Compound3-(5-(bis(2-sulfoethyl)carbamoyl)-2-((1E,3Z,5E)-5-(5-(bis(2-sulfoethyl)carbamoyl)-3,3-dimethyl-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate[C11]

3-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)propanamide (5 mg, 20 μmol)is combined with the succinimidyl ester of fluorochrome F11 (15 μmol)and 10 μL of N-methylmorpholine in 200 μL of anhydrous DMF in a sealedreaction tube and rotated at 37° C. for 2 h. The crude product isprecipitated by addition of 1.5 mL of ethyl acetate, then purified byprep HPLC and the mass confirmed by LCMS.

Example 16: Synthesis of Nonbinding Control Compound3-(2-((1E,3Z,5E)-3-(5-((6-(benzylamino)-6-oxohexyl)carbamoyl)pyridin-2-yl)-5-(1,1-dimethyl-6,8-disulfo-3-(3-sulfopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-3-yl)propane-1-sulfonate[D3]

Benzylamine (1.4 mg, 13 μmol), the succinimidyl ester of fluorochrome F3(11 μmol) and 6.2 μL N-methylmorpholine (55 μmol), were combined in 105μL of anhydrous DMF in a sealed vial and rotated at 25° C. for 1 hour.The crude product was precipitated by addition of 1.5 mL of ethylacetate, centrifuged, decanted and dried under vacuum. The product waspurified by HPLC and the mass confirmed by LCMS: Calculated for [M+H]+1342.2, found 1342.1.

Example 17: Imaging of Tumors In Vivo Using CA Agents

This example shows that the compounds of the invention can be used toimage tumors in vivo. In this experiment, NU/NU mice 6-8 weeks old(Charles River Laboratory, Wilmington, Mass.) were injectedsubcutaneously (s.c.) with about 2×10⁶ HeLa cells bilaterally in eachmammary fat pad. When tumors reached an approximate size of 3×3 mm, micewere injected intravenously (i.v.) with 2 nmoles of carbonic anhydraseagent C5 or nonbinding control agent D3 (5 mice/probe+2 mice/no probe ascontrol) in 100 μL volume via tail vein. Imaging was conducted at 24 hrspost-injection using a Fluorescence Molecular Tomography system (VisEnMedical, Bedford, Mass.) that is commercially available. Examples ofimages using are depicted in FIG. 1, which shows that the tumors can beimaged effectively by both planar reflectance (1A) imaging andtomographically (1B) in vivo with agent C5 while the nonbinding controlagent D3 shows little or no tumor signal by either imaging mode.

Example 18: Affinity of CA Agents to Carbonic Anhydrase In Vitro

This example shows that the compounds of the invention have highaffinity for carbonic anhydrases and are selective for carbonicanhydrase IX over CA II, CA XII and CA XIV in vitro. Also shown is thatthe control compound D3 does not have high affinity to carbonicanhydrases. In this experiment, K_(i) values were determined bymeasuring the rate of CO₂ hydration of the CA spectrophotometrically bya stopped flow method in the presence of varying concentrations of theCA agents. The results are summarized in Table 5.

TABLE 5 K_(I) (nM) Compound CA II CA IX CA XII CA XIV B6 (Example 2) 5029.9 40 69 A1 (Example 8) 288 8.4 57 77 A5 (Example 9) 271 7.9 43 45 A3(Example 11) 9.8 7.6 36 57 A6 (Example 10) 27.1 7.7 40 84 D3 (Example16) >100000 >100000 >100000 >100000

Table 5 depicts the K_(i) values for each experiment, which demonstratethat the CA agents exhibit stronger affinity for carbonic anhydrase IXcompared to CA II, CA XII and CA XIV while no specific binding isdetected for the control compound D3.

Example 19: Carbonic Anhydrase Dependent Binding of CA Agents to HeLaCells

This example shows the affinity of the carbonic anhydrase agentsdescribed herein for CA IX. Cell binding assays using HeLa cellscultured under both hypoxic conditions (1% 02) were performed. Helacells (28,000 cells/cm²) were cultured in 6 well culture plates for 24 hunder normoxic (air) or hypoxic (1% 02) conditions. Incubation with afluorescent anti CA IX Ab (R&D systems) was performed in one well as apositive control followed by incubation for 1 h at 37° C. Carbonicanhydrase agents A5 and A3 (1 μM) or nonbinding control agent D3 wereadded to individual wells followed by incubation for 30 min. Cells wererinsed three times with PBS then scraped with 2 mL, transferred to 5 mLtubes and then spun for 10 min at 1000 rpm to discard the PBS. The cellswere then re-suspend the cells in 400 μL PBS and analyzed byfluorescence microscopy and flow cytometry. For fluorescence microscopy,nuclei were co-stained with DAPI. For the competition study, cells wereincubated with acetazolamide (Az) (100 μM) for 2 hour before incubationwith agents.

FIG. 2 shows that compounds A5 and A3 bound to CA IX receptors on thesurface of the hypoxic cells but not normoxic cells and the binding canbe blocked with an excess of the CA inhibitor acetazolamide. FIG. 2 alsoshows that the commercial fluorescent CA IX antibody binds only tohypoxic HeLa cells confirming upregulation and expression of CA IX inthese cells, and that the nonbinding control agent D3 doesn't bind toeither normoxic or hypoxic cells. These results demonstrate thatcompounds A5 and A3 bind to hypoxic cells in a carbonicanhydrase-dependent manner. FIG. 3 shows flow cytometry resultsconfirming increased binding of the CA agents A5 and A3 but notnonbinding control compound D3 to hypoxic cells and blocking of thesignal with acetazolamide.

Example 20: Biodistribution of I.V. Administered Compound C5 in Mice

This example shows the biodistribution of compound C5 in HeLa tumorbearing mice 24 h post i.v. injection.

Female NU/NU mice 6-8 weeks old (Charles River Laboratory, Wilmington,Mass.) were injected subcutaneously (s.c.) with HeLa cells (5×10⁶) inthe first mammary fat pads. Once tumors reached the desired volume(measured with calipers using the formula volume mm³=(length×width)/2),mice were injected i.v. with compound C5 (2 nmoles) or nonbindingcontrol D3. Mice were sacrificed by carbon dioxide asphyxiation 24 hrlater and certain tissues removed, rinsed with saline, blotted dry, andthe imaged on a VisEn FMT 2500 using the reflectance mode. Regions ofinterest (ROIs) were drawn around each organ using the FMT Software andthe mean fluorescence (reported as counts/energy) determined for eachorgan and normalized to the mean fluorescence in tumors taken to be100%. The results are summarized in FIG. 4, which shows that CA imagingagent C5 fluorescence is very low in most tissues with the highconcentrations found in tumors, and lower concentrations accumulated inthe kidneys while the nonbinding control D3 shows fluorescenceaccumulation only in the kidneys.

Example 21: Effect of Carbonic Anhydrase Inhibitor AcetazolamideTreatment on Carbonic Anhydrase Signal in HeLa Tumor-Bearing Mice

This example demonstrates the effects of carbonic anhydrase inhibitortreatment on carbonic anhydrase HeLa tumor-bearing mice.

HeLa tumor-bearing mice were randomized once their tumors reached thedesired volume and injected i.v. with either C5 (2 nmoles). Forcompetition studies, mice were injected with the acetazolamide (10mg/kg) followed 60 minutes later by compound C5 (2 nmoles). Imaging wasperformed 24 hours after administration of the agents using the FMT2500. As depicted in FIG. 5A, the acetazolamide treated mice had reducedsignal of the exemplary agent C5 compared to untreated animals.

As depicted in FIG. 5B, CA agent C5 showed significant accumulation intumors in vivo, (116 pmol as quantified by FMT) while the acetazolamidetreated animals had approximately 66% less signal (39 pmol as quantifiedby FMT) demonstrating carbonic anhydrase specificity of the in vivotumor signal of the CA imaging agents described herein.

Example 22: Detection of In Vivo Induction of Hypoxia with FluorescentCarbonic Anhydrase Binding Compound in Mice with HeLa Tumors

Female nu/nu mice at 4-5 weeks old, obtained from (Harlan Laboratory,Indianapolis, Ind.) were injected subcutaneously (s.c.) with HeLa cells(1.5×10⁶ cells/site) in the mammary fat pads. Once tumors reached thedesired volume of 600-700 mm³ (measured with calipers using the formulavolume mm³=length×width²/2), mice were grouped randomly (n=4-5mice/group) for with some exposed to controlled hypoxic environmentalconditions (8% oxygen) in a chamber with monitored oxygen levels.Control mice with matched tumor volumes were maintained under normalhousing conditions. C5, (2 nmol) was injected intravenously to all miceat 24 hours after the initiation of the experiment. Hypoxic mice wereplaced back into the hypoxic chamber after the injection, and tumorswere excised and imaged by fluorescence reflectance imaging (FRI) 24hours following injection. FIG. 6 shows fluorescence images of theexcised tumors from mice that were placed in normal air (left) and 8% 02(right). Quantitative analysis of the images is shown in the graphindicating significantly greater uptake of C5 in tumors from mice underhypoxic atmosphere.

Example 23: Distribution of Fluorescent Carbonic Anhydrase BindingCompound in Tumor Tissue by Fluorescence Microscopy

FIG. 7 shows the distribution of C5 fluorescence signal in HeLa tumorbearing mice as determined by fluorescence microscopy of tumor tissuesections co-stained with anti CA IX antibody or pimonidazole (a hypoxiamarker). Tumor bearing mice injected with C5 were then injectedintravenously with pimonidazole (80 mg/kg), a marker of hypoxic tissue,and Hoechst 33342 (25 mg/kg), which stains highly perfused tissue, onehour and five minutes before sacrifice, respectively (38). Tumors,kidneys and muscle tissues were collected and imaged by FRI. The tissueswere then embedded, frozen in OCT (Optimal Cutting Temperaturecompound), and stored at −80° C. until they were sectioned forfluorescence microscopy and immunostaining. Sections of 8 μm thicknesswere prepared and air dried for 10 min. Sections were imaged for Hoechst(blue) in combination with C5 (red). After acquiring images, thesections were fixed in ice-cold acetone for 20 min and incubated inSuperBlock (37515, Thermo Scientific) for 30 min. The tissue sectionswere then stained with detection antibodies diluted with blockingsolution for 1 hour; CA IX expression was detected withfluorescein-conjugated Anti-Human CA IX monoclonal antibody (FAB2188F,R&D System) diluted 1:10 and pimonidazole binding was detected byFITC-conjugated murine anti-pimonidazole monoclonal antibody (providedin the Hypoxyprobe plus kit) diluted 1:25 to a adjacent section. Thesections were imaged again in blue (Hoechst) and green (pimonidazole andCA IX) fluorescence. Finally, the sections were stained with H&Eaccording to standard protocol. Images were taken by Carl Zeiss Axiovertfluorescence microscope. All images were taken at 25× magnification(2.5× objective) with identical exposure times. The tumor tissuedistribution of C5 co-localizes with both CA IX antibody and the hypoxiamarker pimonidazole and is located away from well perfused areasindicated by the Hoechst stain.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications cited herein arehereby expressly incorporated by reference in their entirety and for allpurposes to the same extent as if each was so individually denoted.Further carbonic anhydrase imaging agents are described in U.S. Pat. No.7,833,737; and International Application Publication Nos. WO2006/137092,WO2008/124703, WO 2010/147666, and WO2010/065906, all of which areincorporated herein by reference in their entirety.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A carbonic anhydrase targeting agent represented by formula (I), or asalt thereof:

wherein: CAB is a carbonic anhydrase binding moiety comprising asulfonamide and an aliphatic, aromatic or heteroaromatic moiety; Q is asubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group;or Q is absent; L₁, L₂, and L₃ each represent independently for eachoccurrence a bond or a linker moiety; W represents a benzo-condensedring; X is, independently for each occurrence, C(CH₂Y₁)(CH₂Y₂), O, S, orSe; Y₁ and Y₂ are independently hydrogen or a C₁-C₂₀ aliphatic group,each of which is optionally substituted with L₃-M; M, independently foreach occurrence, is hydrogen or a chemical modifying moiety.
 2. Acarbonic anhydrase targeting agent represented by formula (II), or asalt thereof:

wherein: CAB is a carbonic anhydrase binding moiety comprising asulfonamide and an aliphatic, aromatic or heteroaromatic moiety; Q is asubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, or thioalkyl group;or Q is absent; L₁, L₂, and L₃ each represent independently for eachoccurrence a bond or a linker moiety; R is, independently for eachoccurrence, hydrogen, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, C₁-C₁₈ alkyl, alkenyl, alkynyl, alkoxy, orthioalkyl group, each of which is optionally substituted with L₃-M; n is1, 2, or 3; W represents a benzo-condensed ring; X is, independently foreach occurrence, C(CH₂Y₁)(CH₂Y₂), O, S, or Se; Y₁ and Y₂ areindependently hydrogen or a C₁-C₂₀ aliphatic group, each of which isoptionally substituted with L₃-M; and M, independently for eachoccurrence, is hydrogen or a chemical modifying moiety.
 3. The agent ofclaim 1, wherein the agent is fluorescent in the far-red ornear-infrared.
 4. The agent of claim 1, wherein Q is unsubstitutedheteroaryl.
 5. The agent of claim 1, wherein Q is alkyl.
 6. The agent ofclaim 1, wherein each of L₁, L₂, and L₃ independently comprises—NH—(CH₂)_(n)—C(═O)— where n=1-8, or a diradical of a moiety selectedfrom the group consisting of glycine, alanine, β-alanine,4-aminomethylbenzoic acid, cysteic acid, glutamic acid,amino-polyethylene glycol-carboxylic acid, amino-polyethylene glycolamine, ethylenediamine, propylenediamine, spermidine, spermine,hexanediamine, a diamine-amino acid, lysine, ornithine, diaminobutyricacid, diaminopropionic acid, succinic acid, glutaric acid, suberic acid,adipic acid, amide, triazole, urea, and thiourea.
 7. The agent of claim1, wherein L₁ is one of the following: (a) —C(O)N(R¹)-alkyl-C(O)N(R¹)-ω;(b) —C(O)N(R¹)-alkyl-ω; (c)—C(O)N(R¹)_(z)—C₂₋₃alkyl-O]_(z)—C₁₋₃alkyl-C(O)N(R¹)-ω; (d)—C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω; (e)—C(O)N(R¹)-alkyl-C(O)N(R¹)—C₁₋₃alkyl-aryl-C(O)N(R¹)-ω; or (f)—C(O)N(R¹)—[C₂₋₃alkyl-O]_(z)—C₁₋₃alkyl-C(O)N(R¹)-alkyl-aryl-C(O)N(R¹)-ω;wherein z is an integer from about 3 to about 35, R¹ representsindependently for each occurrence hydrogen or alkyl, and ω is a bond toCAB.
 8. The agent of claim 1, wherein L₂ is C₁₋₅alkyl.
 9. The agent ofclaim 1, wherein L₃ is a bond.
 10. The agent of claim 1, wherein X isC(CH₃)₂.
 11. The agent of claim 1, wherein M represents independentlyfor each occurrence a sulfonate or —SO₃H.
 12. The agent of claim 1,wherein M represents independently for each occurrence a substituentselected from the group consisting of a carboxylate, phosphonate,phosphate, and iminodiacetate.
 13. The agent of claim 1, wherein M is asulfonate.
 14. The agent of claim 1, wherein M represents independentlyfor each occurrence a substituent selected from the group consisting ofhydrogen, alcohol, sulfonic acid, sulfonate, polysulfonate, cysteicacid, sulfonamide, sulfoxide, sulfone, carboxylate, ketone, phosphonate,phosphate; iminodiacetate, ethylenediamine tetra-acetic acid,diethylenetriamine pentaacetic acid, tetra-azacyclododecane tetra-aceticacid, an amino acid or polyamino acid, oligo- or polyethylene glycol,amine, quaternary ammonium ion, a sugar, glucosamine, galactosamine,mannosamine, polyethylene glycol (PEG), alkoxy polyethylene glycol, abranched polypropylene glycol, polypropylene glycol, a graft copolymerof poly-lysine and methoxypolyethyleneglycol, a peptide, a lipid, afatty acid, palmitate, phospholipid, a phospholipid-PEG conjugate, acarbohydrate, an iron oxide nanoparticle, naphthylalanine,phenylalanine, 3,3-diphenylpropylamine, taurine, a phosphonate, aphosphate, a carboxylate, and a polycarboxylate.
 15. The agent of claim1, wherein the chemical modifier(s) M enhance the binding selectivity ofthe agent for CA IX relative to a CA other than CA IX.
 16. The agent ofclaim 1, wherein the M modified agent has a net negative charge rangingfrom −3 and −12 at neutral pH.
 17. The agent of claim 1, wherein thechemical modifier(s) M reduce the nonspecific cell membrane permeabilityof the agent.
 18. The agent of claim 1, wherein the chemical modifier(s)M reduce the nonspecific tissue accumulation of the compound whenadministered to a live animal.
 19. (canceled)
 20. The agent of claim 1,wherein CAB is -aryl-SO₂NH₂, -heterocyclyl-SO₂NH₂,-aryl-C(O)N(R′)-heteroaryl-SO₂NH₂,-heteroaryl-C(O)N(R′)-heteroaryl-SO₂NH₂,—C(O)N(R′)-heteroaryl-heteroaryl-SO₂NH₂, or—C(O)N(R′)-aryl-heteroaryl-SO₂NH₂, wherein R′ represents independentlyfor each occurrence hydrogen or C₁₋₆alkyl; each aryl and heteroaryl areoptionally substituted with 1 or 2 substituents independently selectedfrom the group consisting of alkyl and halogen, and each heterocyclyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of alkyl, halogen, amino, and oxo.21. (canceled)
 22. (canceled)
 23. (canceled)
 24. The agent of claim 1,wherein CAB is represented by


25. The agent of claim 1, wherein the carbonic anhydrase targeting agentis3-(2-((1E,3Z,5E)-5-(3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)-3-(5-((3-oxo-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)propyl)carbamoyl)pyridin-2-yl)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1-sulfonate;and pharmaceutically acceptable salts thereof.
 26. A pharmaceuticalcomposition suitable for administration to a subject, comprising theagent of claim 1 and a pharmaceutically acceptable excipient. 27-44.(canceled)